Sequential hybridization workflow from common lysate

Abstract

A method includes inserting a fluid including a sample into a cartridge, lysing the sample in an extraction chamber to release nucleic acids of interest from the sample, collecting a first set of hybridized magnetic particles hybridized to a first type of nucleic acids of interest by: hybridizing the first type of nucleic acids of interest with an oligonucleotide attached to magnetic particles, holding the first set of hybridized magnetic particles on a side of the extraction chamber, suspending the first set of hybridized magnetic particles with a wash buffer, transporting the first set of hybridized magnetic particles to a detection chamber, collecting a second set of hybridized magnetic particles hybridized to a second type of nucleic acids of interest, amplifying the sets of hybridized magnetic particles, and detecting a plurality of amplification products indicative of the presence, absence, or amount of the types of nucleic acids of interest.

Description

Atty. Dkt. No.: 86357089SEQUENTIAL HYBRIDIZATION WORKFLOW FROM COMMON LYSATE BACKGROUND

[0001] A presence of nucleic acids (e.g., DNA and RNA) in a biological sample or specimen may be useful in diagnosing patients. For example, a presence or amount of a certain nucleic acid may indicate infection or other illness or disease. Preparation of the biological sample may allow the biological sample to be amplified so that an increased number of the nucleic acids proportional to the starting amount of nucleic acids can be detected. Amplification of the nucleic acids may be performed through a method such as a polymerase chain reaction. Detection of the nucleic acids may indicate a presence, absence, or amount of the nucleic acid present in the biological sample.SUMMARY

[0002] This disclosure relates to devices and apparatus for detecting or measuring the presence, absence or amount of a nucleic acid of interest (e.g., a target nucleic acid), in a sample containing or suspected of containing the nucleic acid of interest.

[0003] One aspect relates to a method for detecting a presence, absence, or amount of a nucleic acid of interest. The method includes inserting, into a cartridge, a fluid including a biological sample, wherein the cartridge includes a plurality of zones including an extraction zone including one or more extraction chambers, each including a plurality of magnetic particles, and a detection zone including one or more detection chambers, each detection chamber including one or more heating elements, wherein each zone of the plurality of zones is in fluid communication with each other, and wherein the cartridge further includes one or more reagents, lysing the biological sample into at least one of the one or more extraction chambers to release the one or more nucleic acids of interest from the biological sample, wherein lysing the biological sample produces a lysate, collecting a first set of hybridized magnetic particles, the first set of magnetic particles hybridized to a first type of nucleic acids of interest, by: hybridizing, into the at least one of the one or more extraction chambers, the first type of nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles, the at least one capture oligonucleotide specific to the first type of nucleic acids of interest, holding the first set of hybridized magnetic particles on a side of the extraction chamber, transporting the lysate to a waste storage chamber, suspending the first set of hybridized magnetic particles in the at least one of the one or more extraction chambers with a first wash buffer, and transporting the first set of hybridizedAtty. Dkt. No.: 86357089magnetic particles to a first detection chamber of the one or more detection chambers, collecting, using the lysate, a second set of hybridized magnetic particles, the second set of magnetic particles hybridized to a second type of nucleic acids of interest, amplifying each of the first and second sets of hybridized magnetic particles in at least one of the plurality of zones, via an amplification reaction, to provide a plurality of the first type of the nucleic acid of interest and the second type of the nucleic acid of interest, and detecting a plurality of amplification products indicative of the presence, absence, or amount of the plurality of the first and second types of nucleic acids of interest via an optical unit in communication with each of the first and second detection chambers.

[0004] At least one aspect relates to a diagnostic system. The diagnostic system includes a cartridge including a plurality of zones, in fluid communication with each other, including an extraction zone including: one or more extraction chambers including: a plurality of sets of magnetic particles, wherein the one or more extraction chambers are configured to hold the plurality of magnetic particles, a waste storage chamber configured to receive a lysate from at least one of the one or more extraction chambers, a plurality of wash buffer blisters, each wash buffer blister to wash a different set of the plurality of sets of magnetics particles, and a detection zone including: a plurality of mixing chambers including one or more different reagents, a plurality of detection chambers, a heating element capable of heating the plurality of detection chambers, and a master mix.

[0005] At least one aspect relates to a method for serial hybridization. The method includes: lysing a biological sample into at least one of one or more extraction chambers to release one or more nucleic acids of interest from the biological sample, wherein lysing the biological sample produces a lysate, collecting a first set of hybridized magnetic particles, the first set of magnetic particles hybridized to a first type of nucleic acids of interest, by: hybridizing, into the at least one of the one or more extraction chambers, the first type of nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles, the at least one capture oligonucleotide specific to the first type of nucleic acids of interest, holding the first set of hybridized magnetic particles on a side of the extraction chamber, transporting the lysate to a waste storage chamber, suspending the first set of hybridized magnetic particles in the at least one of the one or more extraction chambers with a first wash buffer, transporting the first set of hybridized magnetic particles to a first detection chamber of the one or more detection chambers, and collecting, using the lysate, aAtty. Dkt. No.: 86357089second set of hybridized magnetic particles, the second set of magnetic particles hybridized to a second type of nucleic acids of interest.BRIEF DESCRIPTION OF THE FIGURES

[0006] These and other aspects and features of the present implementations are depicted by way of example in the figures discussed herein. Present implementations can be directed to, but are not limited to, examples depicted in the figures discussed herein. Thus, this disclosure is not limited to any figure or portion thereof depicted or referenced herein, or any aspect described herein with respect to any figures depicted or referenced herein.

[0007] FIG. 1 A depicts a block diagram of a cartridge and an instrument, in accordance with example implementations.

[0008] FIG. IB depicts a block diagram of a cartridge and an instrument, in accordance with example implementations.

[0009] FIG. 2A depicts a method of serial hybridization, in accordance with example implementations.

[0010] FIG. 2B depicts a method for serial hybridization, in accordance with example implementations.

[0011] FIG. 3 depicts a method for serial hybridization, in accordance with example implementations

[0012] FIG. 4A depicts a first cartridge architecture, in accordance with example implementations.

[0013] FIG. 4B depicts a second cartridge architecture, in accordance with example implementations.

[0014] FIG. 5A depicts a first cross sectional view of a detection chamber of the cartridge of FIGS 1 A-4B, in accordance with example implementations.

[0015] FIG. 5B depicts a second cross sectional view of a detection chamber of the cartridge of FIGS 1A-4B, in accordance with example implementations.Atty. Dkt. No.: 86357089

[0016] FIG. 6 depicts a method for serial hybridization, in accordance with example implementations.

[0017] FIG. 7 depicts a method of pulse-controlled amplification, in accordance with example implementations.

[0018] FIG. 8A depicts a blister device, in accordance with example implementations.

[0019] FIG. 8B depicts the blister device of FIG. 8 A, in accordance with example implementations.

[0020] FIG. 8C depicts the blister device of FIG. 8 A, in accordance with example implementations.

[0021] FIG. 8D depicts the blister device of FIG. 8 A, in accordance with example implementations.

[0022] FIG. 9A depicts a blister device, in accordance with example implementations.

[0023] FIG. 9B depicts an actuator of the blister device of FIG. 9A, in accordance with example implementations.

[0024] FIG. 10A depicts a system, in accordance with example implementations.

[0025] FIG. 10B depicts a system, in accordance with example implementations.

[0026] FIG. 10C depicts a system, in accordance with example implementations.

[0027] FIG. 11A depicts a cartridge environment, in accordance with example implementations.

[0028] FIG. 11B depicts a cartridge environment, in accordance with example implementations.

[0029] FIG. 12A depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.

[0030] FIG. 12B depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.Atty. Dkt. No.: 86357089

[0031] FIG. 12C depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.

[0032] FIG. 12D depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.

[0033] FIG. 13 A depicts a cartridge environment in plan view, in accordance with example implementations.

[0034] FIG. 13B depicts a cartridge environment in plan view, in accordance with example implementations.

[0035] FIG. 14 depicts a cartridge panel, in accordance with example implementations.

[0036] FIG. 15 depicts a user interface for cartridge environment, in accordance with example implementations.

[0037] It will be recognized that the figures are schematic representations of examples for purposes of illustration. The figures are provided for the purpose of illustrating example implementations with the explicit understanding that the figures will not be used to limit the scope of the meaning of the claims. Thus, the description is not limited to the examples and / or implementations provided in the drawings.DETAILED DESCRIPTION

[0038] Biological samples may contain molecules or particles that are of interest. For example, the molecules may be indicative of disease, illness, genetic abnormalities, etc. Molecular diagnostics may be of use to accurately diagnose potential infections or other diseases. Currently, molecular diagnosing may be performed, at least partially, manually. For example, preparing a sample to be analyzed for diagnostic purposed may be performed manually by a human. Manual sample preparation may be slow. Further, a skilled technician may be required to perform the sample preparation. The preparation may be required to be performed in a certified laboratory meeting certain standards or conditions. This may be costly and timeconsuming.

[0039] Further, molecular detection may be performed by amplifying a molecule of interest (also referred to as a “target” or “target molecule”) in the biological sample and detecting aAtty. Dkt. No.: 86357089presence, absence, and / or amount of the molecule of interest. In various examples, the molecule of interest may be a nucleic acid. Specifically, the target molecule may be an oligonucleotide (a “target oligonucleotide”). The target oligonucleotide may be a nucleic acid of interest that is present in and extracted from the biological sample. The target oligonucleotide may be single stranded or double stranded (i.e., before denaturation). The target nucleic acid sequence is the sequence that is amplified. This specific nucleic acid sequence may characterize the presence of a pathogen (e.g., a virus or bacteria) for which the diagnostic method is being used.

[0040] One method of amplification may be isothermal amplification (e.g., the entire reaction or reaction chamber is heated and cooled to a uniform temperature). Isothermal reactions may be associated with low-plex reactions (e.g., reactions having a limited number of targets or components), high costs for reagents used in the reactions, and less sensitivity and / or specificity compared to other types of amplification reactions, such as polymerase chain reactions (PCR). Further, sample preparation methods, specifically for magnetized particles used for amplification reactions, may include additional steps. For example, a chaotropic salt / alcohol sample preparation method may include air drying alcohol and eluting a target molecule from the magnetic particles. These additional steps may incur additional costs and may increase an amount of time it takes for diagnosing. An extraction-free sample preparation may be limited by a reaction volume, as high costs of reagents may limit volumes that can be used for a given reaction or analysis. A limited reaction volume may subsequently limit an amount of target analytes that can reach the amplification reaction step(s), thus reducing sensitivity of the results.

[0041] The systems and methods described herein provide rapid, low-cost, point-of-care molecular diagnostics. The systems and methods may be convenient to both health care providers running the diagnostics and patients waiting to be diagnosed. The systems and methods described herein utilize pulse-controlled amplification (PCA) to amplify and subsequently detect molecules of interest that may be found in a biological sample. PCA reactions may enable rapid thermocycling with a low power expenditure by using paramagnetic particles to concentrate DNA or other nucleic acid targets into a thin thermocycling zone. Additionally, PCA utilizes polymerase chain reaction (PCR) chemistry to replicate and amplify target molecules (e.g., nucleic acids of interest, such as DNA or RNA). The paramagnetic particles may automate and integrate solid-phase extraction of the sample specimen with PCA and a real-time multi-channel detection, thus reducing both a run time of the diagnostics and aAtty. Dkt. No.: 86357089cost of running the diagnostics. Additionally, the diagnostics are performed in a low-cost, disposable cartridge that provides an automated sample-to-answer process. Further, the systems and methods described herein reduce power consumption of instruments used to carry out a multiplex reaction by utilizing low power, yet solid-state and rapid, thermocycling for PCR.

[0042] In one example, a cartridge can include a plurality of zones. The cartridge may be inserted into an instrument that helps to facilitate amplification and detection of the nucleic acid of interest. Different zones of the plurality of zones of the cartridge may include different components and / or have different functionalities. Each of the plurality of zones and / or components may be fluidly coupled and / or may fluidly communicate with one another. This communication may facilitate the steps of the method (e.g., lysing, hybridization, washing, amplification, detection, etc.) to be performed in or on the cartridge to detect and / or analyze nucleic acids of interest, thus reducing a time to reach a diagnosis. For example, a first zone of the plurality of zones may include an input chamber to input a biological sample containing the nucleic acids of interest. A second zone may include one or more extraction chambers, and a third zone may include one or more detection chambers. Additional zones may include, for example, storage for liquid and / or dry reagents (e.g., a wash buffer, a system composition, lyophilized reagents, etc.). A zone may also be used for reconstitution of a master mix used during amplification. The different zones may be coupled via fluid channels. The cartridge can be inserted into an instrument when the process is ready to begin, and the cartridge may only need to be removed from the instrument upon completion of the processes without any external fluid or reagents being added to the process.

[0043] The cartridge may further include valves (e.g., membrane pinch valves, wax valves, volcano valves, etc.) to permit fluid flow from zone to zone, chamber to chamber, etc. in such a way that fluids and reagents are delivered to the appropriate locations within the cartridge at appropriate times / during appropriate steps of the processes. Further, an instrument interfacing with the cartridge may include a plurality of pumps (e.g., syringe pumps, air cylinder pumps, diaphragm pumps, etc.) to allow fluid flow through the cartridge. Thus, each step that occurs in the process of detecting a molecule of interest, from input of the sample to detection of the amplified molecules, may be performed on the cartridge. Further, all reagents, reactants, and other components used during the processes may be stored in and disposed of on the cartridge. The cartridge used to perform the processes herein may be a single use cartridge. Thus, eachAtty. Dkt. No.: 86357089component (e.g., reagents) needed for the various reactions and processes may be stored on the cartridge. Wet and / or dry reagents may be stored in blister devices having frangible seals and / or valves that can control flow of the liquids. Further, used or discarded liquids can be transported back to an original storage blister to reduce a need for a waste chamber in the cartridge or removal of waste external to the cartridge.

[0044] Furthermore, the systems and methods described herein utilize PCA reactions, which may be PCR-like amplifications. The amplification may be able to be performed rapidly (e.g., within 5 to 10 minutes), thus reducing a total time it takes for a sample to be analyzed and a diagnosis to be made. Further, the systems and methods described herein leverage chemistry associated with PCR reactions for a multiplex reaction (e.g., a reaction that facilitates detecting multiple nucleic acids of interest) and real-time detection. Multiplex reactions allow for the ability to detect and amplify multiple nucleic acids of interest. Further, multiplex reactions may allow for detection and amplification of different types of nucleic acids of interest simultaneously (e.g., while the nucleic acids are in the same chamber or different chambers of the cartridge). This may allow for diagnosis of multiple diseases, infections, etc. with one biological sample, thus reducing time, costs, and labor associated with diagnostics.

[0045] As previously stated, real-time detection may reduce analysis times and a time to make a diagnosis based on the analysis results. Further, solid-phase extraction may enhance a sensitivity of the results, though purification and concentration. Solid-phase extraction may also allow more challenging specimen types to be analyzed. Specificity of capture of the target molecules may also be increased through hybridization of the target molecules.

[0046] The systems and methods described herein utilize pulse-controlled amplification to amplify molecules (e.g., nucleic acids) of interest that may be found in a biological sample. The biological sample may be lysed (e.g., with a system composition and / or a control substance) to isolate the molecules of interest from the biological sample. In some examples, the molecules of interest are nucleic acids and may be referred to as “nucleic acids of interest.” The nucleic acids of interest may also be referred to herein as “target oligonucleotides,” each of which may have a unique oligonucleotide sequence. The isolated molecules of interest may be hybridized to magnetic particles having attached capture oligonucleotides. The capture oligonucleotides may refer to the oligonucleotides attached to the particles, each having a nucleic acid sequence that is complementary to the “target oligonucleotide.” A capture oligonucleotide may be an oligonucleotide attached to the magnetic particles having a sequenceAtty. Dkt. No.: 86357089designed to be complementary to the “target oligonucleotide.” The capture oligonucleotide may be a synthetic single-stranded segments of nucleic acid (DNA or RNA). The sequence of the capture oligonucleotide may be designed specifically to match (i.e., complement) the nucleic acid sequence of the target oligonucleotide. In various examples, the capture oligonucleotides may be further used to isolate and amplify the nucleic acid of interest. However, it should be understood that various other methods can be used to capture nucleic acids of interest and subsequently isolate and amplify the nucleic acids of interest in proximity to a heating element.

[0047] Hybridization may occur by annealing the molecule of interest with the attached capture oligonucleotides having a complementary sequence. The hybridized molecules of interest and magnetic particles may be amplified to generate an increased or amplified number of molecules of interest via a PCA reaction. The presence, absence, and / or amount of the molecule of interest may be determined using, for example, optical detection.

[0048] High-plex PCA amplification may be difficult to perform successfully. For example, off-target amplification, uneven amplification, etc., may prevent analytes of interest from being sufficiently amplified such that all analytes of interest can be successfully detected. Additionally, division of the biological sample or lysate (e.g., into aliquots) during sample preparation may reduce sensitivity during detection. For example, dividing the biological sample or lysate into three aliquots prior to detection may reduce a detection limit by three times, making detection of an analyte difficult. Detection may be performed using high Stokeshift fluorescent dyes or temperature-differentiated fluorescent probes, which may address limitations of optical fluorescence detection. Melting curve analysis may also be performed. However, methods may not address challenges posed by high-plex PCA amplification (e.g., practical limits to a number or probes or channels in a detection system may still exist). For example, high Stokes-shift dyes are limited by a number of channels in a fluorescence detection system and have higher associated costs compared to standard dyes. Further, a melt curve analysis using molecular beacons may be costly in terms of running time. Melting curve analysis is also challenging with PCA reactions due to variability, and is limited by a number of channels in the detection system. Further, running sample preparations multiple times, either with a larger amount of the biological sample or using a shared lysed sample, can address the challenges posed by high-plex PCA amplification. However, these methods can increase a size, complexity, and cost of the workflow.Atty. Dkt. No.: 86357089

[0049] Specifically, the systems and methods described herein provide detection of a plurality of analytes (e.g., nucleic acids of interest) in a multiplex or high-plex reaction to address the challenges described above. A high-plex reaction may be a type of multiplex reaction in which a large (e.g., above a certain threshold value) number of analytes are analyzed from a single biological sample. A large number of analytes may be, for example, eight or more analytes. To enable analysis of a large number of analytes, the systems and methods described herein utilize multiple sequential low-plex hybridization, wash, and particle capture workflows (referred to as “serial hybridization workflows”) . Each serial hybridization workflow may utilize the same (e.g., a common lysate). Each hybridization workflow, as will be described herein, includes, at a high level, hybridizing nucleic acids of interest to magnetic particles, washing the hybridized magnetic particles, and capturing the magnetic particles in a detection chamber. Once each sample (e.g., hybridized magnetic particle) is captured in a detection chamber, a PCA reaction is performed to amplify the nucleic acids of interest. The amplified nucleic acids of interest are then detected. Amplification and detection may be performed in parallel or series (e.g., amplification and detection of each serial hybridization workflow product is performed in parallel or series). Amplification of each sample may be a low or lower-plex reaction, thereby facilitating an overall high-plex reaction.

[0050] Serial hybridization workflows may be associated with numerous technical advantages. As discussed above, a sample or lysate is not split, which would reduce sensitivity. Further, a number of analytes is not limited by detection system limitations. For example, a standard (e.g., 6-channel, 6-target) optical detection system may be able to be utilized by using one such detection system for each sample. Further, the number of analytes is not limited by PCA reaction limitations, as any n number of samples can be amplified and detected. Further, multi-(e.g., six-) plex amplification may be able to be performed with real-time detection. Additionally, as described herein, a common heating element (e.g., foil assembly) may be shared across all detection chambers in the cartridge. A common heating element may reduce an added cost of additional reaction or detection chambers and heating elements.

[0051] Referring now to Fig. 1A, a diagnostic system 100A, comprising a cartridge 100 and an instrument 150 is shown, according to an example implementation. The system 100A may be configured to detect a nucleic acid of interest. In some examples, the cartridge 100 of the diagnostic system may include a series of zones or chambers that may be defined as cavities. The zones / chambers / cavities may be interconnected via a plurality of channels. The series ofAtty. Dkt. No.: 86357089cavities / chambers can be individually loaded with reagents. The cavities / chambers can be loaded with the same or with different types of reagents. Loading the cavities / chambers with different types of reagents can permit a multi-step reaction. The reagents / reactants can be disposed in the chambers or blister devices, as will be described herein.

[0052] As will be described herein, the cartridge 100 includes a plurality of zones, each in fluid communication with each other. The extraction zone 104 includes one or more extraction chambers 114. Each of the one or more extraction chambers 114 includes a plurality of sets of magnetic particles 116. The one or more extraction chambers 114 may hold or store the plurality of sets of magnetic particles 116.

[0053] As shown in FIG. 1 A, the cartridge 100 includes a first zone, referred to as an extraction zone 104, comprising a first extraction chamber 114 having magnetic particles 116 and reagents 125, a first wash buffer 110a housed in a first blister device 126a, a second wash buffer 110b housed in a second blister device 126b, and a waste storage chamber 168. The cartridge 100 may also include a detection zone 108, including a first detection chamber 120a, a second detection chamber 120b, and a heating element 122 that is common or shared among both detection chambers 120. The instrument 150 includes an optical unit 162.

[0054] The cartridge 100 may also include a plurality of zones, shown as the extraction zone 104 and the detection zone 108. Each zone may be or include a plurality of chambers. For example, the extraction zone 104 may include an extraction chambers 114 and the detection zone 108 may include the detection chambers 120. Each zone of the plurality of zones may be in fluid communication with each other (e.g., the other zones of the plurality of zones). It should be understood that the cartridge 100 may include any number of zones. In various examples, each zone is configured to perform or is associated with certain actions.

[0055] Fluid within the cartridge 100 may move through a plurality of fluidic channels. The channels may be fluidly coupled via a plurality of fluidic junctions. Transport of fluid through the cartridge 100 may be moderated by a plurality of valves. For example, the cartridge 100 may include a plurality of pinch valves, membrane valves, etc. configured to selectively permit and restrict flow through the fluidic channels. Membrane pinch valves may be specific types of valves used to restrict and permit fluid flow through the cartridge. Valves may also be different types of valves, such as wax valves or volcano valves. The use of valves is described in greater detail with respect to FIGS. 4A and 4B.Atty. Dkt. No.: 86357089

[0056] For example, when a valve is closed, fluid movement may be restricted. The fluidic channels may permit fluid flow from one of the plurality of zones and / or chambers to another zone and / or chamber. For example, fluid may flow from the extraction zone or an extraction chamber to a detection zone or detection chambers. In various examples, the instrument 150 may include a pump to interface with the cartridge 100, specifically the fluid channels of the cartridge 100, to pump air to move air and the fluid through the channels. For example, the instrument 150 may include pump fluidic interconnect used to pump air, fluid, etc. through the cartridge 100. A pump may be, for example, a syringe pump, an air cylinder pump, a diaphragm pump, etc. The use of a pump in the cartridge 100 is described in greater detail with respect to FIGS. 4A and 4B. In various examples, a pressure differential across each valve opening can be applied to transport fluid in and out of each of the plurality of zones, each of the plurality of chambers, each blister device, etc.

[0057] The cartridge 100 may include a greater or fewer number of zones than what is shown in FIG. 1A. For example, the cartridge 100 may include first, second, third, and fourth zones, each housing different components, chambers, and / or elements of the cartridge 100. In various examples, different elements of the cartridge 100 may be included in different zones, depending on the configuration of the cartridge 100. For example, as shown in FIG. 1 A, the cartridge 100 may include the extraction zone 104 having the extraction chambers 114, the wash buffers 110, and the waste storage chamber 168, and the detection zone 108 having the detection chambers 120 and the heating element 122. In FIG. IB, the detection zone 108 further includes a plurality of mixing chambers 123, each including reagents 125.

[0058] In various examples, at least one of the plurality of extraction chambers 114 may be an extraction chamber 114. The extraction chambers 114 may house a lysing process of the biological sample to release one or more nucleic acids of interest from the biological sample. The extraction chamber 114 may be a fluidic structure with an open cavity or void to define a chamber that could be filled with the biological sample comprising the one or more nucleic acids of interest. The extraction chamber 114 may be configured to house a lyse reaction. For example, a biological sample may enter the extraction chamber 114 and may be lysed, for example by sonication, thermal lysis, thermal sonication, or another lysing method. The lyse reaction may damage the content of the biological sample to release one or more nucleic acids from the biological sample. The lyse reaction performed in the extraction chamber 114 mayAtty. Dkt. No.: 86357089cause the one or more nucleic acids of interest to be released from the biological sample. The lyse reaction may disrupt, or lyse, cells, and / or tissue samples.

[0059] As shown in FIG. 1 A, the extraction zone 104 may include a first wash buffer 110a and a second wash buffer 110b. The extraction zone 104 further includes a plurality of wash buffer blisters, shown as wash buffer blisters 126a and 126b. Each wash buffer blister 126a and 126b may suspend a different set of the plurality of sets of magnetic particles 116 within the extraction chamber 114. It should be understood that the wash buffers 110 may be included in a zone different than the extraction zone 104. In various examples, the wash buffers 110 are not included in a zone. The system 100 A may include one or more wash buffers 110a. In various examples, the cartridge 100 may include a chamber in a first zone of a plurality of zones to store the wash buffer 110. The wash buffer 110 may be transported from the chamber to at least one extraction chamber 114 and / or detection chamber 120 to remove undesired elements of the biological sample when the biological sample is in the chamber. In various examples, the extraction chamber 114 and / or the detection chamber 120 may be washed to remove undesirable elements from the chamber being washed.

[0060] The wash buffers 110 refers to buffers used for washing magnetic particles 116, the extraction chamber 114, and / or the detection chamber 120. In some examples, the wash buffers 110 can comprise water, a salt, a buffering compound or component (e.g., tris buffer) and / or a surfactant. In some examples, the salt is KC1, MgCh NaCl, etc. In some examples, the wash buffer 110 comprises between 20mM and 45mM salt. In some examples, the wash buffer can comprise between about 0.001% and about 0.1% (e.g., about 0.001%, 0.01%, 0.05%, or 0.1%) surfactant. In some examples, the surfactant is selected from Tween ®20, Tween® 80, Tween® 85, SPAN® 80 or SPAN® 85. In some examples, the surfactant is Tween® 20.

[0061] The system 100A may include a plurality of wash buffers 110. Each wash buffer 110 may have a different composition (e.g., one buffer can be or include salt while another buffer can be or include water). For example, a first wash buffer 110a may have a more aggressive wash chemical relative to a second wash buffer. A more aggressive wash chemical may be a harsher chemical that can remove a greater number of undesired elements in the sample of fluid relative to a less aggressive wash chemical, such as because the more aggressive wash chemical can have a higher chemical concentration, pH level, a higher specificity for removing the undesired elements, etc. Thus, the first wash buffer may be used to wash a chamber and the second wash buffer may be used to the chamber. In some examples, the wash buffers may beAtty. Dkt. No.: 86357089used to suspend and / or resuspend the hybridized magnetic particles 116 within the extraction chamber 114. For example, the first wash buffer may be introduced to a chamber (e.g., the detection chamber 120, the extraction chamber 114, etc.) to wash or remove undesired elements of the sample. Undesired elements may be, for example: remaining sample fluid that is not the nucleic acid of interest, contaminants, or other elements or molecules that may interfere with use of the extracted nucleic acid of interest. The second wash buffer may be introduced to the chamber after the first wash buffer has exited the chamber and the wash has been completed. The rinse may remove any remaining elements or contaminants not removed by the first wash buffer.

[0062] The system 100A may include any number of wash buffers 110. A number of wash buffers 110 may correspond to a number of serial hybridizations that may occur within the extraction chamber 114. For example, wash buffer stored in a blister device may be used for only one hybridization.

[0063] The cartridge 100 may also include one or more reagents 125. The reagents 125 may be located in at least one of the one or more extraction chambers. For example, as shown in FIG. 1 A, the extraction chamber 114 includes one or more reagents 125, however, it should be understood that the reagents 125 may be stored in any chamber or zone and / or multiple chambers or zones of the cartridge 100. For example, as shown in FIG. IB, the reagents 125 are stored in the mixing chambers 123a and 123b. In various examples, the cartridge 100 may include only one reagent in only one chamber (e.g., the detection chamber 120). In various other examples, no reagent 125 may be stored in any extraction chamber 114 or detection chamber 120, but may be stored elsewhere in the cartridge 100. For example, the reagents may reside in any extraction or detection chambers, but may be stored elsewhere instead.

[0064] The one or more 125 may be or include liquid reagents 125 and / or dry reagents 125. The reagents 125 may be used in lysing, hybridization, washing, amplification, and / or detection of the nucleic acids of interest. The reagents 125 may also be or include a dry reagent storing an internal positive control (IPC) organism. The reagents 125 may be or include a master mix reagent, shown in FIG. IB as a master mix 127. The master mix reagent 127 may be stored in the detection zone 108. The master mix (MM) reagent may be or include a master mix lyophilized (“lyo”) bead. The master mix reagent may be used during amplification for reverse transcription and / or amplification and real-time detection of the nucleic acids of interest. TheAtty. Dkt. No.: 86357089reagents 125 may be used in the lyse reaction. For example, an IPC may be used, in addition to a system composition or buffer, to lyse the biological sample.

[0065] As used herein, a “master mix” (“MM”) or “PCR mixture” refers to a mixture of reagents useful for an amplification reaction (e.g., a PCA reaction, an RT-PCA reaction, a PCR reaction, an RT-PCR reaction, a qPCR reaction). In some examples, the master mix may comprise polymerase, dNTPs, primers (e.g., at least a forward and a reverse primer specific for a target), a probe comprising a detectable label (e.g., a fluorescent probe), and / or a reverse transcriptase. In some examples, the dNTPs comprise a detectable label. In some examples, the master mix is 3X concentration (meaning the master mix comprises 3 times the concentration of each amplification ingredient than needed for the amplification reaction, a 3X master mix is reconstituted (diluted) three folds in a PCA buffer), 5X concentration (meaning the master mix comprises 5 times concentration of each amplification ingredient than needed for the amplification reaction, a 5X master mix is reconstituted (diluted) five folds in a PCA buffer), or 10X concentration (meaning the master mix comprises 10 times the concentration of each amplification ingredient than needed for the amplification reaction, a 10X master mix is reconstituted (diluted) ten folds in a PCA buffer). In some examples, the master mix is lyophilized. In some examples, the master mix is lyophilized in a lyoprotectant, such as trehalose. In some examples, a lyophilized master mix is reconstituted in the PCA buffer as described herein. In various examples, reconstitution of the master mix may be performed by one or more of: reciprocating flow between chambers and / or channels, ultrasonic and / or acoustic mixing, impellers, mechanical agitation, diffusion, and / or channel geometry (e.g., turns, ridges, mini-chambers, etc.). In various examples, one or more components or liquid reagents 125 (e.g., the system composition and / or the wash buffer 110) may be stored, housed, or otherwise contained in a blister device 126. Example blister devices are described in greater detail with respect to FIGS. 8A-9B. The blister devices 126 may store liquid reagents (e.g., the system composition, the wash buffer 110, liquid reagents used for amplification, etc.). The blister device 126 may allow fluid to be released so that the fluid can reach a destination (e.g., the detection chamber 120). In various examples, the cartridge 100 may be configured such that the system composition and / or the wash buffer 110 may return to the blister device 126 upon completion of use. For example, the wash buffer 110 may be released from a blister device 126 storing the wash buffer 110 and may be delivered to the detection chamber 120 to wash the contents of the detection chamber. After washing is complete, the wash buffer 110 may return to the blister device 126 to be stored.Atty. Dkt. No.: 86357089

[0066] In various examples, liquid reagents 125 may be stored in blister devices (e.g., blister devices 126 of FIGS. 1A and IB) of the cartridge. For example, liquid reagents (e.g., wash buffer, system composition) may be stored in a metal-lined blister device. Blister devices are described in greater detail with respect to FIGS. 8A-9B. The one or more reagents 125 may be located in at least one of the one or more extraction chambers 114 or at least one of the one or more detection chambers 120.

[0067] The blister device 126 can be configured to transport its contents in a direction according to a selection of a pumping direction through the blister device 126. In some examples, the blister device 126 is configured to transport contents of the blister device 126 in a direction from a first valve toward a second valve. In some other examples, the blister device 126 is configured to transport contents of the blister device 126 in a direction from the second valve toward the first valve. Blister devices may also be referred to herein as a blisters. The blister device(s) 126, when present in the cartridge 100, may be or be part of the first zone and / or the second zone of the plurality of zones.

[0068] The blister device 126 may comprise a chamber (e.g., a storage cavity) to store a reagent, a first actuator at a first end of the zone, and / or a second actuator at a second end of the zone opposite to the first end of the zone. The blister device 126 can be configured to be hermetically sealed-off from the channel(s) of the cartridge 100 when the diagnostic device / system is in a non-activated state.

[0069] In various examples, dry reagents 125 may be stored as lyophilized (e.g., freeze-dried) pellets or cakes, air-dried pellets or cakes, and / or sealed with a plastic plug or film. For example, a dry reagent may be an enzyme used for DNA or RNA elongation during amplification. The dry reagent can be or include master mix or a PCR mixture. The dry reagent 125 may be stored as a pellet. Dry reagents may be dissolved in order to be properly utilized. In various examples, a device may push against a piston to push a liquid out of a blister device to a location of a pellet to dissolve the pellet. The dissolved pellet may then be transported to a desired location (e.g., the detection chamber 120).

[0070] The extraction zone 104 may also include the waste storage chamber 168. The waste storage chamber 168 receives a lysate from at least one of the one or more extraction chambers 114. For example, as described herein, upon lysing the biological sample, a lysate may beAtty. Dkt. No.: 86357089moved to the waste storage chamber 168 so that the lysate does not contact any of the detection chambers 120 (e.g., or the detection zone 108).

[0071] In some examples, the extraction chamber 114 includes a heating system. The extraction chamber 114 may be heated to a specific temperature to release the desired molecule of interest from the biological sample (e.g., the nucleic acid of interest). In some other examples, the heating extraction zone or chamber may be a serpentine channel where a fluid of interest having the biological sample is heated during the fluid passage. A serpentine channel may include a series of U-shaped channels that alternate in direction. A serpentine channel may increase a distance the fluid flowing through the channel travels. This may allow ample time to head the fluid to a target temperature.

[0072] In various examples, the biological sample can be lysed by a different lyse system (e.g., the lyse system 152, shown in FIG. IB), which may be located within the instrument 150. In various examples, the lysate can be lysed by a plurality of lysing techniques, such as a combination of the lyse system 152, mechanical agitation, an external heat source, ultrasonic agitation, impellers, and / or ceramic or glass beads. In various examples, the lyse system may include a sonicator (shown in FIG. IB as sonicator 154) to interface with the one or more extraction chambers of the cartridge 100. The sonicator may be used for lysing. The sonicator 154 will be described in greater detail with respect to FIG. IB. The sonicator may also be referred to as a “sonotrode.”

[0073] In various examples, at least one of the plurality of extraction chambers 114 may be an extraction chamber 114. The extraction chamber 114 may include a plurality of magnetic particles 116. The plurality of magnetic particles 116 may include one or more capture oligonucleotides that may be complementary to the one or more nucleic acids of interest. The capture oligonucleotides may be complementary to the nucleic acids of interest because the capture oligonucleotides may be configured and / or selected to bind specifically to (e.g., and only to) the one or more nucleic acids of interest. In the extraction chamber 114, the one or more nucleic acids may be hybridized. For example, in the extraction chamber 114, the one or more nucleic acids of interest may be hybridized with at least one capture oligonucleotide attached to one or more magnetic particles 116 of the plurality of magnetic particles.

[0074] The term “hybridize” as used herein refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of atAtty. Dkt. No.: 86357089least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs. For example, hybridization may refer to the formation of a double strand from two single strands, which can each include a nucleic acid and / or a capture oligonucleotide. The capture oligonucleotide may be, for example a DNA or RNA sequence having a complementary sequence to the nucleic acid of interest. Under suitable reaction conditions, the hybridization generally leads to the lowest possible energy state that can be achieved by the combination of the two single strands. In other words, under suitable conditions, the two single strands may bind to each other in such a way that, with respect to the sequences of the two single strands, the greatest possible complementarity (i.e., specificity) is produced.

[0075] In some examples, hybridizations are conducted with probe-length nucleic acid molecules, 15-100 nucleotides in length, or 18-50 nucleotides in length. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point I of the formed hybrid. The stringency of hybridization conditions may be estimated and / or adjusted such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. In some examples, specific hybridization occurs under stringent hybridization conditions. An oligonucleotide or polynucleotide (e.g., a probe or a capture oligonucleotide) that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions.

[0076] As described herein, a plurality of hybridization may be performed. The hybridizations may be performed in series (e.g., serial hybridization). A different hybridization may be performed for each different type of nucleic acid of interest to be amplified and detected. In various implementations, each nucleic acid of interest (e.g., and associated magnetic particles 116) may have a different associated lysate. In some examples, all nucleic acids of interest have and / or use the same lysate.

[0077] The cartridge 100 may include a plurality of the magnetic particles 116. Specifically, the extraction chamber 114 may include a plurality of the magnetic particles 116. The magnetic particles 116 may also be referred to as “magnetic beads,” “beads,” and / or “particles” As used herein, “magnetic beads” or “magnetic particles” refer to microparticles that haveAtty. Dkt. No.: 86357089ferromagnetic or paramagnetic properties. For example, the magnetic particles 116 may be or include (strept)avidin with biotinylated oligonucleotides, covalently bound oligonucleotides, polymer beads with embedded iron particles, etc. The size of the microparticles is optionally in a range from approximately 10 nm to approximately 2 mm, optionally in a range from 100 nm to 1 mm, optionally in a range from 500 nm to 50 pm. The shape of the microparticles can be freely selected and can, for example, be spherical, cube-shaped, cuboid, or ellipsoidal. The magnetic particles with ferromagnetic properties are optionally formed from at least one of the following materials or contain at least one of the following materials: iron, nickel, cobalt, AlNiCo, SmCo, Nd2Fei4B, NieoFe2o (“Permalloy”), and / or NiFeCo alloys. Optionally, the magnetic particles with paramagnetic properties are made of at least one of the following materials formed or contain at least one of the following materials: alkaline earth metals, alkali metals, and / or rare earths. Alternatively, a magnetic microparticle can be formed from a nonmagnetic material such as glass and / or silicate, with magnetic substances being embedded therein. For example, such a microparticle can have a core made of magnetic materials. The magnetic microparticles are optionally provided with at least one coating in order to enable or promote functionalization with nucleic acids, in particular with extraction nucleic acids and / or capture oligonucleotides. Optionally, at least one extraction nucleic acid and / or one capture oligonucleotides and a maximum of 1012extraction nucleic acids and / or capture oligonucleotides are functionalized on a microparticle. Optionally, an areal density of extraction nucleic acids and / or capture oligonucleotides that are functionalized on the surface of a magnetic microparticle is in a range from 0.0001 to 1 per square nanometer. The microparticles can optionally have a coating which enables and / or facilitates functionalization with capture oligonucleotides. For example, the surface of the magnetic microparticles can be at least partially functionalized with streptavidin.

[0078] The magnetic particles 116 may have a nucleic acid attached. Specifically, the attached nucleic acids may be attached to the magnetic particles 116 by capture oligonucleotides that are attached to the magnetic particles 116. The capture oligonucleotides attached to the magnetic particles 116 are selected to be complementary in part or in full to the one or more nucleic acids of interest. In the extraction chamber 114, the one or more nucleic acids from the extraction chamber 114 may be hybridized to the capture oligonucleotides attached to the magnetic particles 116. In some examples, the magnetic particles 116 comprise one or more capture oligonucleotides that bind to the one or more nucleic acids of interest (e.g., from the extraction chamber 114). In various examples, the capture oligonucleotides may be the sameAtty. Dkt. No.: 86357089or different from each other. For example, a first capture oligonucleotides attached to a first magnetic particle 116 may have a first sequence, and a second capture oligonucleotides attached to a second magnetic particle 116 may have a second sequence. That is, the magnetic particles 116 may include any number of sets of magnetic particles. Each set of magnetic particles may have features (e.g., different capture oligonucleotides) that cause different nucleic acids of interest to be hybridized with the magnetic particles. For example, the magnetic particles 116 may include 1, 2, 3, 4, 5, etc. sets of magnetic particles 116. Each set may be associated with a different or common lysate within the extraction chamber 114 and a different set of nucleic acids of interest.

[0079] In some examples, the plurality of magnetic particles 116 include one or more of: oligonucleotides capable of capturing different nucleic acids of interest or different types of magnetic particles, each type of magnetic particle having a different oligonucleotide capable of capturing a specific nucleic acid of interest.

[0080] In various examples, the cartridge 100 includes a plurality of extraction chambers 114. Lysing and hybridization may both occur in the same extraction chamber 114. During hybridization, the fluid containing the biological sample be mixed (e.g., partial mixing, complete mixing, etc.) during hybridization. Mixing the fluid may occur to distribute the magnetic particles 116 throughout the fluid (e.g., the lysate). Mixing may additionally or alternatively occur using one or more of ultrasonic mixing, acoustic mixing, impellers, mechanical agitation, diffusion, and / or channel geometry, such as turns, ridges, and / or mini chambers.

[0081] During hybridization, the fluid may also be heated. The solution may be heated in a variety of ways, such as using an external contact heat source, ultrasonic energy, acoustic energy, and / or infrared radiation (IR).

[0082] The detection zone 108 may be, in various examples, a second zone including one or more detection chambers 120. In various examples, a different zone may include the one or more detection chambers. The detection chambers 120 may not be a part of or associated with a zone.

[0083] The cartridge 100 may include a plurality of detection chambers 120 (e.g., a first detection chamber 120a and a second detection chamber 120b). The detection chambers 120 may be configured to amplify and detect the one or more nucleic acids of interest that haveAtty. Dkt. No.: 86357089been lysed (e.g., from the biological sample) and hybridized (e.g., to the functionalized magnetic particles 116). Thus, in various examples, at least one of the one or more detection chambers 120 is an amplification chamber. The detection chambers 120 may amplify the nucleic acid of interest through, for example, pulse controlled amplification (PCA). PCA is described in greater detail with respect to FIG. 7.

[0084] The cartridge 100 may include a number of detection chambers 120 corresponding to a number of serial hybridizations performed. That is, different sets of magnetic particles 116 may be associated with different nucleic acids of interest. Each set of magnetic particles 116 may be amplified in a different detection chamber.

[0085] The detection zone 108 may also include a plurality of detection chambers 120, shown as detection chamber 120a and detection chamber 120b in FIG. 1 A and referred to collectively as detection chamber or chambers 120. The detection zone 108 may include a heating element (e.g., a heating element 122) capable of heating the plurality of detection chambers. As shown in FIG. 1A, the heating element 122 may heat any or all of the detection chambers 120. As shown in FIG. IB, each detection chamber 120 may include an individual heating element 122.

[0086] As used herein, the terms “amplify” or “amplification” with respect to nucleic acid sequences, refer to methods that increase the representation of a population of nucleic acid sequences in a sample. Copies of a particular target nucleic acid sequence generated in vitro in an amplification reaction may be referred to as “amplicons” or “amplification products.” In various examples, amplification products may refer to any products amplified during an amplification reaction. For example, amplification products may include nucleic acids, unquenched fluorophores generated during each replication cycle of the amplification reaction, etc. Amplification may be exponential or linear. A target nucleic acid may be DNA (such as, for example, genomic DNA and complementary DNA (cDNA) or RNA). While the methods described hereinafter relate to amplification using polymerase chain reaction (PCR), numerous other methods such as isothermal methods, rolling circle methods, etc., may be used either in place of, or together with, PCR methods. In at least one of the detection chambers 120, a lyophilized master mix reagent may be stored. The master mix reagent may refer to a reagent used during an amplification reaction, such as a PCR or PCA reaction.

[0087] In various examples, the amplification reactions may utilize primers to perform amplification of the nucleic acids. Primers are short, artificial, single-stranded segments ofAtty. Dkt. No.: 86357089nucleic acid (e.g., DNA) that are designed to be complementary to the beginning and / or end of the target sequence that will be amplified. The primer sequences may be shorter than the one of the capture oligonucleotides. For example, a primer may contain about 10 to 25 nucleotides.

[0088] Primers may perform specific functions during amplification. For example, during the amplification / elongation step of the PCR, the primers may bind to both ends of the nucleic acid of interest (e.g., the DNA sequence of interest), thus bookending the sequence of interest that need to be amplified. Enzymes (e.g., DNA polymerase) may then copy the part of the target oligonucleotide sequence that falls between the primers, selectively amplifying the sequence of interest. In some examples, the capture oligonucleotides described above may also be used as primers during amplification. In other examples, the primers may be oligonucleotides different than the capture oligonucleotides. In some examples, the primers may be forward and / or reverse primers. Forward and reverse primers may denote a direction of elongation during the polymerization by the polymerase enzyme. The primers may be used during the amplification / elongation step of the reaction and may comprise part of the master mix composition described herein.

[0089] The detection chambers 120 may include a heating element 122. For example, a detection zone of a plurality of zones may include one or more detection chambers 120 having one or more heating elements 122. Each detection chamber 120 may include a heating element 122. In some examples, the heating element 122 may be common or shared among all detection chambers 120. In various examples, at least one of the one or more detection chambers may be an amplification chamber. Further, the heating element of the amplification chamber may be a foil configured to interact with electrical components of the instrument 150. The electrical components may provide an electrical connection between the instrument 150 and the detection chambers 120, thus providing heat modulation to the detection chamber 120 to amplify the one or more nucleic acids of interest that have been hybridized to the capture oligonucleotides of the plurality of magnetic particles. Specifically, the electrical connection may activate the one or more heating elements 122 of each of the one or more detection chambers 120.

[0090] The heating element 122 may be positioned or located at a side portion (e.g., only a single side, such as a single wall of the detection chamber 120) of the detection chambers 120. The heating element 122 may include a plurality of layers comprising at least a foil. The heating element 122 may be attached to one or more of an adhesive and / or a heat spreader or conductor. The heating element 122 may be or include, in various examples, a continuous, structured, orAtty. Dkt. No.: 86357089shaped metal foil, metal wires, a conductor and / or resistor layer deposited and / or plated, and / or backed by a heat spreader. The heating element 122 may be configured to heat the detection chambers 120 during the PCA process. The heating element 122 may be, for example, a foil to locally heat a portion of the detection chambers 120. For example, a heating element 122 of the amplification chamber (e.g., the detection chamber 120) may be or include a foil to interact with electrical components of the instrument 150 provide an electrical connection to activate the one or more heating elements 122 of each of the one or more detection chambers 120. The heating element 122 may provide heat modulation to amplify the one or more nucleic acids of interest that have been hybridized to the at least one capture oligonucleotide attached to one or more magnetic particles 116 of the plurality of magnetic particles in the extraction chamber 114. The detection chamber 120 is described in greater detail with respect to FIGS. 5A and 5B.

[0091] During the PCA reaction occurring in the detection chamber 120, the heating element 122 may be used to heat a portion of the volume of the detection chamber 120. For example, only a portion of the detection chamber 120 proximate the heating element 122 may be heated. The heating element 122 may be heated by a short electric pulse or other process. Each pulse may be short (e.g., between about 10 and about 1200 microseconds) and each pulse may be delivered quickly in succession. In some examples, the heating time in at least one amplification cycle, in at least 10, at least 20, at least 40, at least 80, or in at least 160 amplification cycles is more than 1 nanosecond, more than 5 nanoseconds, or more than 10 nanoseconds and less than 100 milliseconds, less than 10 milliseconds, less than 1 millisecond, less than 300 microseconds, less than 100 microseconds, less than 50 microseconds, less than 30 microseconds, less than 10 microseconds, less than 5 microseconds, or less than 1.5 microseconds.

[0092] Due to a resistance of the foil and the rapid pulsing, heat created by the pulses may be local (e.g., the pulses heat only a small portion of the detection chamber 120 surrounding the heating element). In various examples, the pulses may be generated by an energy or power. When the pulses are ceased or removed, the portion of the detection chamber 120 that has been heated by the pulses may return to the same temperature as the rest of the detection chamber not proximate the heating element 122. As such, denaturation of the nucleic acids or other molecules occurs in a small volume near the heating element 122.

[0093] Denaturation of a nucleic acid may include separate it into its two single strands. For example, the original can be separated from the complement during denaturing. DenaturingAtty. Dkt. No.: 86357089may also be referred to as melting. The denaturing of the nucleic acid double strand may be thermally induced. For example, at least a part of the nucleic acid double strand or the whole double strand is exposed to a temperature, described as a denaturing temperature, which causes or at least encourages a separation of the nucleic acid double strands. The denaturing temperature may not be a fixed temperature but may be a temperature interval, within which the temperature during denaturing varies. The denaturing temperature may be selected to be so high that nucleic acid double strands can be separated and / or so low that a DNA polymerase, which may aid in denaturation, is not substantially damaged. In some examples, a denaturation temperature may be between 90 and 100 degrees Celsius. For example, a denaturing temperature may be 95 degrees Celsius.

[0094] This heating process may cause the overall temperature of the detection chamber 120 to be isothermal (e.g., no change, substantially no change, or minimal change in temperature is seen during heating for the PCA reaction), because the heated portion of the detection chamber 120 is small enough that an overall temperature change is not seen by the detection chamber 120. In various implementations, heat flow in and out of the detection chamber 120 may be present. The heating element 122 may cause a slight (e.g., less than 1 degree Celsius) increase in the overall temperature of the detection chamber 120. For example, heat may be unable to be transferred out of the detection chamber 120, and each pulse generated by the heating element 122 may add heat to the chamber. However, the pulse may heat a small enough volume of the detection chamber 120 relative to the total volume of the detection chamber 120 such that the volume heated by the heating element 122 can rapidly return to a setpoint temperature of the detection chamber 120.

[0095] Referring still to FIG. 1A, the instrument 150 is shown, according to example implementations. The diagnostic system 100A may include the instrument 150 to interface with (e.g., connect to) the cartridge 100. The cartridge 100 may be inserted into the instrument 150 to perform the processes described herein. In various examples, the cartridge 100 may be a single use cartridge. For example, one cartridge may be used one time to detect the presence of nucleic acids in one biological sample.

[0096] The instrument 150 may include an optical unit 162. The optical unit 162 may be coupled with at least one of the one or more detection chambers. The optical unit 162 may be configured to detect a presence, absence, amount, etc. of the nucleic acid of interest after amplification has been performed. In various examples, the optical unit 162 is to detect aAtty. Dkt. No.: 86357089plurality of amplification products indicative of a presence, absence, or amount of the amplified one or more nucleic acids of interest. The optical unit 162 may detect the nucleic acid via a label bound to the nucleic acid of interest during amplification.

[0097] In various examples, the cartridge 100 may include a transparent window in at least one detection chamber (e.g., the detection chamber 120) of the one or more detection chambers to allow the optical unit 162 to detect a plurality of amplification products indicative of a presence, absence, or amount of the plurality of amplified nucleic acids of interest. In various examples, the detection chamber 120 may include the transparent window. A transparent window may permit light from the optical unit 162 to pass through the cartridge 100 and / or detection chamber 120 so that light can enter the detection chamber 120 and detect the amplification products indicative of a presence, absence, or amount of the plurality of amplified nucleic acids of interest.

[0098] In some examples, detection by the optical unit 162 may be performed in the detection chamber 120 and may be done using a label bound to the nucleic of interest. Specifically, the optical unit 162 may be used for fluorescence sensing. For example fluorophores may be generated during each replication cycle of the amplification reaction. Fluorophores may be used to infer the presence or amount of nucleic acids in the detection chamber 120. In PCA (and / or PCR), fluorophores may be attached to capture oligonucleotides and a quencher molecule prior to amplification. This compound molecule may be included in the master mix reagent. In the proximity of the quencher, the compound molecule may not fluoresce. However, during PCA (and / or PCR), the compound molecule may anneal or attach to a portion of the target nucleic acids. A polymerase enzyme, while constructing a double stranded amplicon, may release the fluorophore to separate the fluorophore from the quencher and allow the fluorophore to fluoresce.

[0099] In some examples, the detection step may include detecting the fluorophore via a sensor located within the optical unit 162. For example, the optical unit 162 and any sensors located within may be positioned in the instrument 150 such that the optical unit 162 and / or the sensors interface with and / or are located proximate the detection chamber 120 when the cartridge 100 is inserted into the instrument 150. In some examples, the sensor may be a fluorescence sensor coupled to the optical unit 162 and configured to detect the fluorescence from one or more of the plurality of zones of the cartridge 100.Atty. Dkt. No.: 86357089

[0100] In some examples, the optical unit 162 may detect the nucleic acid of interest by detecting an intensity of fluorescent probes in the detection chamber 120. The detection chamber 120 may be configured to transmit fluorescent emission to a sensor within the optical unit 162 located in the instrument. For example, during amplification, fluorophores may be released (e.g., using Taqman probes) into solution during elongation for specific detection of amplified products. The optical unit 162 may detect the fluorophores that may be indicative of the presence, absence, amount, etc. of the nucleic acid of interest.

[0101] Referring now to FIG. IB, a diagnostic system 100B is shown, according to an example implementation. The system 100A includes a cartridge 100 and an instrument 150. The cartridge 100 and the instrument 150 may include the same or similar elements as the elements found in the cartridge and instrument of the system 100A. For example, the extraction chamber 114, and the detection chamber 120 may be the same as those described with respect to FIG.1A.

[0102] The cartridge 100 can include a sample input chamber. The sample input chamber may be a chamber or cavity in which a biological sample can be inserted so that the biological sample enters the cartridge 100 and can be analyzed. For example, the cartridge 100 may be configured to receive a biological sample. The sample input chamber may include a liquid port for receiving liquids. The biological sample may be, for example, saliva, blood, cells, etc. In various examples, the biological sample may include one or more nucleic acids. Of the one or more nucleic acids, the biological sample may include one or more nucleic acids of interest. The nucleic acids of interest may be nucleic acids that the cartridge 100 is being used to amplify and analyze. In various examples, the nucleic acids of interest may not be included in a biological sample. For example, the nucleic acids of interest may be isolated and input into the sample input chamber . The nucleic acids of interest may be, for example, DNA, RNA, mRNA, etc. The one or more nucleic acids of interest may be the same targets or different targets (e.g., the cartridge 100 can be used to detect and / or analyze one or more of the same type of nucleic acid of interest or different nucleic acids of interest). For example, the diagnostic system 100A of 100B may be configured to amplify multiple nucleic acids of interest to, for example, detect multiple infections, diseases, etc. Amplification and identification of these multiple nucleic acids may be performed simultaneously, thus reducing an amount of time and cost of detecting the presence of multiple nucleic acids. The sample input chamber is shown and described in greater detail with respect to FIGS. 12A-D.Atty. Dkt. No.: 86357089

[0103] The sample input chamber may include a sample input cap, which may be a cap that interfaces with a liquid port of the cartridge. The sample input cap may seal the sample input chamber by preventing liquid from exiting the sample input chamber through the liquid port. The sample input chamber may receive a biological sample or other fluid containing one or more nucleic acids of interest. Thus, the cartridge 100 may include one or more sample fill indicators to indicate a fill level of the sample input chamber. The sample fill indicators may be lines, tick marks, or other visual indicators of a volume of liquid or fluid in the sample input chamber. The sample input chamber may include a transparent material to view a volume of fluid in the sample input chamber.

[0104] The system composition may be a buffer or composition useful for lysis and hybridization of a sample (e.g., of the biological sample). The system composition may also be referred to as a “system buffer.” The system composition may include ingredients (e.g., a buffer) to control a pH of the solution. In some examples, the system composition can comprise water, a salt and / or a surfactant. In some examples, the salt is MgCh, NaCl, KC1, or (NH^SCh. In some examples, the system composition comprises between 50mM and IM salt. In some examples, the system composition comprises between about 0.001% and about 0.1% (e.g., about 0.001%, 0.01%, 0.05%, or 0.1%) surfactant. In some examples, the surfactant is selected from Tween® 20 (polysorbate 20), Tween® 80 (polysorbate 80), Tween® 85 (polysorbate 85), SPAN® 80 (sorbitan monooleate) or SPAN® 85 (sorbitane trioleate). In some examples, the surfactant is Tween® 20. The system composition may also include a buffering component to buffer the pH of the solution. For example, the buffering component may be a tris buffer.

[0105] In various examples, the biological sample may be lysed with the system composition and an internal positive control (IPC). The resulting solution of the biological sample, the system composition , and the IPC may be referred to as a lysate. The IPC may be included in the lysate to control false negative results. During amplification, the IPC may be amplified with the nucleic acids of interest to indicate that the solution being amplified is functional and a negative result (e.g., the nucleic acids of interest are not detected) is reliable. The IPC may be a lyophilized organism or synthetic organism. In various examples, the IPC may be a protein or organism present in a human sample. Further, the extraction chamber 114 may include a plurality of beads (e.g., glass beads, ceramic beads) used to mechanically agitate or lyse the organisms present in the lysate.Atty. Dkt. No.: 86357089

[0106] During hybridization, the fluid may also be heated. The solution may be heated in a variety of ways, such as using an external contact heat source, ultrasonic energy, acoustic energy, and / or infrared radiation (IR).

[0107] The cartridge 100, shown in FIG. IB, may include a plurality of mixing chambers 123a and 123b. Specifically, the detection zone 108 includes a plurality of mixing chambers 123, shown in FIG. IB as first mixing chamber 123a and second mixing chamber 123b. Each mixing chamber 123 may store or include one or more different reagents 125. The mixing chambers 123 may be configured to mix the lysate and / or hybridized magnetic particles 116 transported from the extraction chamber 114 prior to entering the detection chambers 120. In some examples, the mixing chambers 123 may be used to suspend the magnetic particles 116 in the wash buffers 110.

[0108] The cartridge 100 may include one or more detection chambers 120. The detection chambers 120 may be configured to amplify and detect the one or more nucleic acids of interest that have been lysed (e.g., from the biological sample) and hybridized (e.g., to the functionalized magnetic particles 116). Thus, in various examples, at least one of the one or more detection chambers 120 is an amplification chamber. The detection chambers 120 may amplify the nucleic acid of interest through, for example, pulse controlled amplification (PC A). PCA is described in greater detail with respect to FIG. 7.

[0109] Referring still to FIG. IB, the instrument 150 is shown, according to example implementations. The diagnostic system 100A may include the instrument 150 to interface with (e.g., connect to) the cartridge 100. The components of the instrument 150 may be positioned such that specific components are located proximate to specific corresponding components of the cartridge 100 when inserted into the instrument 150. The cartridge 100 may be inserted into the instrument 150 to perform the processes described herein. In various examples, the cartridge 100 may be a single use cartridge. For example, one cartridge may be used one time to detect the presence of nucleic acids in one biological sample.

[0110] The instrument 150 of FIG. IB may include a lyse system 152, magnetic field generators 156a and 156b, and the optical unit 162.[OHl] The instrument 150 may include a lyse system 152. The lyse system 152 may interface with at least one of the one or more extraction chambers 114 of the cartridge 100. The lyse system 152 may include a sonicator 154 (shown in FIG. IB). The sonicator 154 may interfaceAtty. Dkt. No.: 86357089with the at least one extraction chamber 114 (e.g., the extraction chamber 114). The lyse system 152 may be utilized when the biological sample enters the extraction chamber 114 of the cartridge 100 to lyse the sample and release the nucleic acids of interest. The lyse system 152 may be coupled to the extraction chamber 114.

[0112] In various examples, the lyse system 152 may be a lyse system comprising sonication (e.g., use of ultrasonic energy), thermal lysis, and / or thermal sonication system. In some examples, the lyse system 152 may include a heating system. In examples where the lyse system 152 includes a heating system, the lyse system 152 may heat the extraction chamber 114 to a target temperature (e.g., between 35 and 100 degrees Celsius), such as by heating the heating element of the extraction chamber 114 to the target temperature, to release the desired molecule of interest (e.g., nucleic acid) from the biological sample. For example, the lyse system 152 may heat the extraction chamber 114 to 95 degrees Celsius.

[0113] In some examples, the lyse system 152 is programmable. Thus, a user may be able to control, set, determine, etc. lysing protocol parameters (e.g., using a controller) such as the sample volume, sonication power level, acoustic frequency, and lysing duration. The lyse system 152 may also provide a cooling feature, enabled by a heat exchanging sub-assembly, which may prevent the biological sample from exceeding a maximum set temperature during operation.

[0114] As stated above, the lyse system 152 may include a sonicator 154. The sonicator 154 may interface with the at least one extraction chamber 114 (e.g., the extraction chamber 114). The sonicator 154 may deliver ultrasonic waves to the extraction chamber 114 to lyse the fluid present in the chamber. In some examples, the biological sample may be lysed using a sonication system, by using a sonotrode (e.g., the sonicator 154). The lyse system 152 of the instrument 150 may therefore include an ultrasonic transducer or sonicator 154 that transmits ultrasonic energy to the extraction chamber (e.g., the extraction chamber 114) into the biological sample to cause cell / spore / tissue disruption. Efficient transfer of the ultrasonic energy from the sonicator 154 to the sample within the extraction chamber 114 may be dependent, at least in part, upon maintaining the contact between the transducer tip of the sonicator 154 present in the instrument and the cartridge according to a predetermined force.

[0115] The instrument 150 may include a first magnetic field generator 156a and a second magnetic field generator 156b to generate a magnetic field. The magnetic field may be used toAtty. Dkt. No.: 86357089dock the plurality of magnetic particles 116 to at least one zone of the plurality of zones of the cartridge 100. In various examples, the magnetic field generator 156 is a magnet (e.g., a permanent magnet). The magnetic field generators 156 may be positioned at or proximate the detection chamber 120. The magnetic field generator may be configured to generate a magnetic field such that the magnetic particles 116 that are hybridized with the nucleic acid of interest and / or the capture oligonucleotides are separated from the lysate solution such that the PCA reaction can occur. The magnetic field generators 156 may be a current carrying conductor or other device that may be powered on or activated such that electric charges being moving to create the magnetic field. In various examples, the instrument 150 may be activated or powered on to activate the magnetic field generator.

[0116] The first magnetic field generator 156a is configured to generate a magnetic field to dock the plurality of magnetic particles to an extraction chamber of the one or more extraction chambers. Further, the second magnetic field generator 156b is configured to capture the magnetic particles 116 (e.g., the hybridized magnetic particles 116) to the heating element 122. The magnetic field generator 156b may force the plurality of magnetic particles towards the one or more heating elements 122, capture the plurality of magnetic particles onto the one or more heating elements 122, cause the plurality of magnetic particles to attach to the heating element 122, cause the plurality of magnetic particles to be located within a distance of the heating element 122, etc. In some examples, the magnetic field generator 156a or 156b is a movable magnetic field generator able to interact with the cartridge at one or more detection chambers of the plurality of detection chambers.

[0117] In some examples, the magnetic field generators 156 is configured to generate a variable magnetic field in such a manner that acts on at least a part of the magnetic particles 116 present in the detection chamber and that are linked to the nucleic acid of interest.

[0118] In another example, the variable magnetic field may act on at least a part of the magnetic particles 116 present in the detection chamber 120 in such a manner that the magnetic particles 116 attach to the local heating element 122. Further, the variable magnetic field may be configured to act on the magnetic particles 116 attached to the local heating element 122 in such a manner that they leave the local heating element 122 and are suspended in a reaction solution. This may allow the magnetic microparticles 116 to be optionally attached to and / or repelled from the local heating element 122 multiple times, allowing them to hybridize with additional target nucleic acids in the reaction solution.Atty. Dkt. No.: 86357089

[0119] The magnet or plurality of magnets may be or include a permanent magnet and / or an electromagnet that can be changed in position and / or orientation relative to the reaction container. For example, when using a permanent magnet, the magnetic field may be changed by changing an orientation of the permanent magnet to the detection chamber 120 and / or a distance of the permanent magnet from the detection chamber 120. The direction of the magnetic field can also be changed, for example, by reversing the permanent magnet relative to the reaction container such that, for example, the side of the permanent magnet facing the reaction container changes from the magnetic north pole to the south pole of the permanent magnet or vice versa. When using an electromagnet, for example, the variable magnetic field can be changed by changing the current flow, in such a manner as the current intensity and / or the direction of the current flow. For example, the electromagnet may comprise one or more solenoid coils and optionally a ferromagnetic core. The magnet may be formed on a side of the local heating element 122 facing away from the detection chamber 120. This may offer the advantage that the permanent magnet in this arrangement makes it particularly effective and easy to attract the magnetic microparticles 116 to the local heating element. Alternatively or additionally, the one or more magnets may be changed in position relative to the detection chamber to provide a variable magnetic field in the reaction solution. Alternatively or additionally, several magnets with different polarity can be brought to the detection chamber to achieve a variable magnetic field in the detection chamber.

[0120] Referring now to FIG. 2A, a method 200 for detecting a presence, absence, or amount of a nucleic acid of interest is shown, according to an example implementation.

[0121] At process 202, a fluid comprising a biological sample is inserted into a cartridge (e.g., the cartridge 100). In various examples, the biological sample may include the nucleic acid of interest. In various examples, the biological sample may contain another non-nucleic acid molecule of interest. In various examples, the nucleic acid of interest may be a DNA strand and / or an RNA strand. The cartridge may include a plurality of zones. Each zone of the plurality of zones may be in fluid communication with each other. For example, the plurality of zones may be or include an extraction zone and / or a detection zone in fluid communication with one another. The extraction zone may include one or more extraction chambers 114 and the detection zone may include one or more detection chambers 120. In various examples, each detection chamber 120 may include one or more heating elements 122. The cartridge may further include one or more reagents 125 and a plurality of magnetic particles 116.Atty. Dkt. No.: 86357089

[0122] At process 204, the biological sample is lysed into at least one of the one or more extraction chambers (e.g., extraction chambers 114). Lysing the biological sample may release the one or more nucleic acids of interest from the biological sample. In various examples, the biological sample may be lysed by, for example, sonication (e.g., using the sonicator 154), heating, mechanical agitation, etc. In various examples, the biological sample may be mixed with an IPC and / or a system composition to form a lysate, which may then be lysed to release the nucleic acids of interest from the biological sample. For example, the method can include lysing, in a device, the biological sample including the one or more nucleic acids of interest to release the one or more nucleic acids of interest from the biological sample. For example, the biological sample is lysed by one or more of sonication, thermal lysis, or thermal sonication.

[0123] At process 206, a first set of hybridized magnetic particles are collected. The first set of magnetic particles may be hybridized to a first type of nucleic acid or acids of interest. The processes 208-216 may be performed to collect the first set of hybridized magnetic particles.

[0124] At process 208, the one or more nucleic acids of interest are hybridized into the at least one extraction chamber 114 of the one or more extraction chambers 114. The one or more nucleic acids of interest may be hybridized to or with at least one capture oligonucleotide attached to one or more magnetic particles 116 of the plurality of magnetic particles 116. For example, the extraction chamber 114 may include a plurality of magnetic particles 116. The magnetic particles 116 may include attached capture oligonucleotides. The nucleic acids of interest may be hybridized to the capture oligonucleotides. The capture oligonucleotides may be specific to the first type of nucleic acids of interest. For example, the capture oligonucleotides attached to and / or functionalized with a first set of magnetic particles (e.g., the magnetic particles to be hybridized with the first type of nucleic acids of interest) may have a sequence that is complementary to a sequence of the first type of nucleic acids of interest. This may allow, during hybridization, only the first type of nucleic acids of interest to become hybridized. After the hybridization with the first set of magnetic particles, yet-to-be-targeted nucleotides (e.g., nucleotides not having a sequence complementary to the first set of capture oligonucleotides) may remain unhybridized in the extraction chamber 114. The yet-to-be-targeted nucleotides may be hybridized in a series of n serial hybridizations.

[0125] At process 210, the first set of hybridized magnetic particles (e.g., the first set of nucleic acids of interest hybridized with the at least one capture oligonucleotide specific to the first type of nucleic acids of interest that is attached to the one or more magnetic particles 116) areAtty. Dkt. No.: 86357089held on a side of the extraction chamber 114. For example, the hybridized magnetic particles 116 are held to the side of the extraction chamber 114 in an applied magnetic field (e.g., as generated by the magnetic field generator 156a or 156b).

[0126] At process 212, the lysate is transported to a waste storage chamber (e.g., the waste storage chamber 168). That is, after the hybridized magnetic particles are held to the side of the lyse chamber (at process 210), the lysate without the hybridized magnetic particles included may be transported to the waste storage chamber 168 or lysate storage chamber 468. In some examples, the waste storage chamber 168 may be a lysate storage chamber. The lysate may be stored in the waste storage chamber 168 until a second set of hybridized magnetic particles is collected. The lysate may then be used to hybridize the second set of magnetic particles to a second type of nucleic acid of interest. That is, a common lysate may be used for collection of all sets of hybridized magnetic particles 116.

[0127] At process 214, the first set of hybridized magnetic particles 116 is suspended. The first set of hybridized magnetic particles 116 is suspended in the at least one of the one or more extraction chambers (e.g., the extraction chamber 114). The first set of hybridized magnetic particles 116 is suspended with a first wash buffer (e.g., a wash buffer 110a).

[0128] In some examples, process 214 includes returning the first wash buffer to a storage blister device (e.g., blister device 126a or 424a) after suspending the first set of hybridized magnetic particles. The first wash buffer may be returned to a blister device that originally stored the first wash buffer (e.g., before the wash buffer was transported to the extraction chamber 114 to wash and / or suspend the hybridized magnetic particles).

[0129] When the first wash buffer is returned to the storage blister, the method 200 (e.g., process 214) may include washing the first set of hybridized magnetic particles with a second wash buffer (e.g., wash buffer 110b). The method 200 may then further include suspending the first set of hybridized magnetic particles in the at least one of the one or more extraction chambers 114 via the second wash buffer 110b.

[0130] At process 216, the first set of hybridized magnetic particles 116 is transported from the extraction chamber 114 to a first detection chamber 120a of the one or more detection chambers 120. When only a first wash buffer is used (e.g., only the first wash buffer washes and / or suspends the first set of hybridized magnetic particles), the first wash buffer may transport the first set of hybridized magnetic particles 116 to the first detection chamber 120a.Atty. Dkt. No.: 86357089In examples where the second wash buffer 110b is used (e.g., and the first wash buffer 110a is returned to the storage blister), the first set of hybridized magnetic particles 116 may be transported to the first detection chamber 120a via the second wash buffer 110b.

[0131] In some examples, the method 200 further includes trapping the first set of hybridized magnetic particles 116 within a threshold distance of the one or more heating elements 122 of the at least one detection chamber 120.

[0132] Collecting the first set of hybridized magnetic particles may further include transporting the lysate from the waste storage chamber 168 back to the at least one of the one or more extraction chambers 114. Transporting the lysate back to the extraction chamber 114 may occur after transporting the first set of hybridized magnetic particles to the first detection chamber 120a of the one or more detection chambers 120 and before collecting the second set of hybridized magnetic particles. Transporting the lysate back to the extraction chamber 114 may allow a common lysate to be used to hybridize each type of nucleic acids of interest with a corresponding set of magnetic particles having capture oligonucleotides attached that are specific to that type of nucleic acids of interest.

[0133] At process 218, a second set of hybridized magnetic particles 116 are collected. The second set of hybridized magnetic particles 116 is hybridized to a second type of nucleic acids of interest. The second set of hybridized magnetic particles is associated of the same lysate as the lysate used to collect the first set of hybridized magnetic particles 116. Collecting the second set of hybridized magnetic particles may include or be similar to the processes 208-216 described above.

[0134] For example, collecting the second set of hybridized magnetic particles 116 may include hybridizing, into the at least one of the one or more extraction chambers (e.g., the extraction chamber 114), the second nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles 116 of the plurality of magnetic particles. The at least one capture oligonucleotide may be specific to the second type of nucleic acid of interest. For example, the capture oligonucleotides attached to and / or functionalized with a second set of magnetic particles (e.g., the magnetic particles to be hybridized with the second type of nucleic acids of interest) may have a sequence that is complementary to a sequence of the second type of nucleic acids of interest. This may allow, during hybridization, only the second type of nucleic acids of interest to become hybridized. After the hybridizationAtty. Dkt. No.: 86357089with the second set of magnetic particles, yet-to-be-targeted nucleotides (e.g., nucleotides not having a sequence complementary to the first or second set of capture oligonucleotides) may remain unhybridized in the extraction chamber 114. The yet-to-be-targeted nucleotides may be hybridized in remaining serial hybridizations.

[0135] Collecting the second set of hybridized magnetic particles 116 may also include holding the second set of hybridized magnetic particles on a side of the extraction chamber 114 (e.g., via an applied magnetic field. Collecting the second set of hybridized magnetic particles 116 may include transporting the lysate to the waste storage chamber 168. Collecting the second set of hybridized magnetic particles 116 may include suspending the second set of hybridized magnetic particles in the at least one of the one or more extraction chambers with a third wash buffer. The third wash buffer may be of the same type as the first wash buffer. For example, the cartridge 100 may include a plurality of wash buffer blisters storing the same type of wash buffer. For example, as is shown and described in FIG. 4A, the cartridge of the diagnostic system architecture 400 includes wash buffer blisters 424a and 424b, each of which stores a first type of wash buffer (e.g., the wash buffer 110a). The wash buffer blisters 424c and 424d each store a second type of wash buffer (e.g., the wash buffer 110b). Each set of hybridized magnetic particles that is collected may be washed with the same types of wash buffers (e.g., first and / or second wash buffers 110a and 110b), but each set of hybridized magnetic particles that is collected is washed with fresh wash buffer (e.g., no wash buffer is reused).

[0136] Collecting the second set of hybridized magnetic particles 116 may also include transporting the second set of hybridized magnetic particles to a second detection chamber of the one or more detection chambers. For example, the second set of hybridized magnetic particles 116 may be transported to the detection chamber 120b, while the first set of hybridized magnetic particles 116 is transported to the detection chamber 120a. Collecting the second set of hybridized magnetic particles 116 may also include transporting the lysate from the waste storage chamber 168 back to the at least one of the one or more extraction chambers 114 after transporting the second set of hybridized magnetic particles to the second detection chamber 120b of the one or more detection chambers 120. This may allow the lysate to be used to hybridized a third set of magnetic particles with a third set of nucleic acids of interest (e.g., and any further sets of magnetic particles and sets of nucleic acids of interest).

[0137] After collecting the second set of hybridized magnetic particles, the method 200 may include transferring a reconstituted first master mix reagent to the first detection chamber 120aAtty. Dkt. No.: 86357089of the one or more detection chambers 120. The method 200 may further include delivering a plurality of amplification reagents 125 unique to the first set of hybridized magnetic particles to the first detection chamber 120a. In various examples, the plurality of amplification reagents 125 may include, for example, a lyophilized master mix reagent, an enzyme for use in amplification, etc. The amplification reagents 125 may be located in various locations of the cartridge (e.g., in blister devices 126) and may be transported to the detection chamber 120 prior to amplification. In various examples, the amplification reagents may be stored as dry or wet reagents. Dry reagents may be dissolved prior to use.

[0138] Additionally, the method 200 may include transferring a reconstituted second master mix reagent to a second detection chamber 120b of the one or more detection chambers 120 (e.g., when the second set of hybridized magnetic particles are to be delivered to the second detection chamber 120b of the one or more detection chambers 120). The method 200 may further include delivering a plurality of amplification reagents 125 unique to the second set of hybridized magnetic particles to the second detection chamber 120b.

[0139] It should be understood that the method 200 may include collecting any number n sets of hybridized magnetic particles (e.g., 1, 2, 3, 4, 5, etc. number of sets of hybridized magnetic particles). As such the processes described with respect to processes 208-206 may be performed n number of times with n types of nucleic acids of interest, and n sets of magnetic particles attached to capture oligonucleotide specific to a particular type of nucleic acids of interest. Further, the cartridge 100 may include n number of detection chambers, master mix reagents, and amplification reagents such that each type of nucleic acids of interest are delivered to a separate detection chamber and have or use master mix and amplification reagents during amplification that are unique to the specific type of nucleic acid of interest. For example, when 3 serial hybridizations are performed (e.g., 3 sets of hybridized magnetic particles are collected), the cartridge 100 may include first, second, and third detection chambers 120a, 120b, and 120c. First master mix reagents and amplification reagents may be specific to the first type of nucleic acids of interest and may be delivered to the first detection chamber 120a. Similarly, second master mix reagents and amplification reagents may be specific to the second type of nucleic acids of interest and may be delivered to the second detection chamber 120b, and third master mix reagents and amplification reagents may be specific to the third type of nucleic acids of interest and may be delivered to the third detection chamber 120c.Atty. Dkt. No.: 86357089

[0140] At process 220, the first and second sets of the hybridized magnetic particles may be amplified in at least one of the plurality of zones (e.g., the detection zone 108). The one or more nucleic acids of interest may be amplified via an amplification reaction (e.g., a PCA reaction). The amplification reaction may provide a plurality of the first type of nucleic acids of interest and the second types of the nucleic acids of interest. For example, the first set of hybridized magnetic particles are delivered to a first detection chamber 120a of the one or more detection chambers to be amplified and the second set of hybridized magnetic particles are be delivered to a second detection chamber 120b of the one or more detection chambers to be amplified. Thus, a first amplification reaction is performed at the first detection chamber 120a to amplify the first type of nucleic acids of interest, and a second amplification reaction is performed at the second detection chamber 120b to amplify the second type of nucleic acids of interest.

[0141] In various examples, amplifying the first and second types of nucleic acids of interest may be performed in the detection chambers 120a and 120b by generating a pulse of current via an electrical connection of an instrument 150. The instrument 150 may receive and interact with the cartridge 100. The pulse of current may modulate a temperature proximate the one or more heating elements 122 of the first and second detection chambers 120a and 120b to increase a temperature proximate the one or more heating elements of the first and second detection chambers 120a and 120b to between 90 and 110 degrees Celsius. For example, the temperature may be increased to 100 degrees Celsius. In various examples, only a portion of the first and second detection chambers may be heated by the pulses. In various examples, amplification may be performed by a PCA reaction.

[0142] At process 222, a plurality of amplification products indicative of the presence, absence, or amount of the plurality of the first and second types of nucleic acids of interest may be detected. The amplification products may be detected via an optical unit 162 in communication with each of the first and second detection chambers 120a and 120b. For example, during amplification, the plurality of nucleic acids of interest may be tagged, for example, using fluorophores. The optical unit 162 may detect an amount of fluorophores, which may be indicative of a presence, absence, and / or amount of the nucleic acid of interest in the biological sample.

[0143] Referring now to FIG. 2B, a method 230 for serial hybridization is shown, according to an example implementation. The method 230 may include processes similar to those describedAtty. Dkt. No.: 86357089above with reference to FIG. 2A. For example, as will be described herein, processes 232-246 may be similar to processes 204-218 described above.

[0144] At process 232, the biological sample is lysed into at least one of the one or more extraction chambers (e.g., extraction chambers 114). Lysing the biological sample may release the one or more nucleic acids of interest from the biological sample. In various examples, the biological sample may be lysed by, for example, sonication (e.g., using the sonicator 154), heating, mechanical agitation, etc. In various examples, the biological sample may be mixed with an IPC and / or a system composition to form a lysate, which may then be lysed to release the nucleic acids of interest from the biological sample. For example, the method can include lysing, in a device, the biological sample including the one or more nucleic acids of interest to release the one or more nucleic acids of interest from the biological sample. For example, the biological sample is lysed by one or more of sonication, thermal lysis, or thermal sonication.

[0145] At process 234, a first set of hybridized magnetic particles are collected. The first set of magnetic particles may be hybridized to a first type of nucleic acid or acids of interest. The processes 236-244 may be performed to collect the first set of hybridized magnetic particles. At process 236, the one or more nucleic acids of interest are hybridized into the at least one extraction chamber 114 of the one or more extraction chambers 114. The one or more nucleic acids of interest may be hybridized to or with at least one capture oligonucleotide attached to one or more magnetic particles 116 of the plurality of magnetic particles 116. For example, the extraction chamber 114 may include a plurality of magnetic particles 116. The magnetic particles 116 may include attached capture oligonucleotides. The nucleic acids of interest may be hybridized to the capture oligonucleotides. The capture oligonucleotides may be specific to the first type of nucleic acids of interest. For example, the capture oligonucleotides attached to and / or functionalized with a first set of magnetic particles (e.g., the magnetic particles to be hybridized with the first type of nucleic acids of interest) may have a sequence that is complementary to a sequence of the first type of nucleic acids of interest. This may allow, during hybridization, only the first type of nucleic acids of interest to become hybridized. After the hybridization with the first set of magnetic particles, yet-to-be-targeted nucleotides (e.g., nucleotides not having a sequence complementary to the first set of capture oligonucleotides) may remain unhybridized in the extraction chamber 114. The yet-to-be-targeted nucleotides may be hybridized in a series of n serial hybridizations.Atty. Dkt. No.: 86357089

[0146] At process 238, the first set of hybridized magnetic particles 116 (e.g., the first set of nucleic acids of interest hybridized with the at least one capture oligonucleotide specific to the first type of nucleic acids of interest that is attached to the one or more magnetic particles 116) are held on a side of the extraction chamber 114. For example, the hybridized magnetic particles 116 are held to the side of the extraction chamber 114 in an applied magnetic field (e.g., as generated by the magnetic field generator 156a or 156b).

[0147] At process 240, the lysate is transported to a waste storage chamber (e.g., the waste storage chamber 168). In some examples, the waste storage chamber 168 may be a lysate storage chamber. The lysate may be stored in the waste storage chamber 168 until a second set of hybridized magnetic particles is collected. The lysate may then be used to hybridize the second set of magnetic particles to a second type of nucleic acid of interest. That is, a common lysate may be used for collection of all sets of hybridized magnetic particles 116.

[0148] At process 242, the first set of hybridized magnetic particles 116 is suspended. The first set of hybridized magnetic particles 116 is suspended in the at least one of the one or more extraction chambers 114. The first set of hybridized magnetic particles 116 is suspended using a first wash buffer (e.g., a wash buffer 110a). Washing may be performed by introducing the wash buffer 110a into the at least one extraction chamber 114. For example, the wash buffer 110a may be released from a blister device 126 and may be transported to the extraction chamber 114.

[0149] At process 244, the first set of hybridized magnetic particles 116 is transported from the extraction chamber 114 to a first detection chamber 120a of the one or more detection chambers 120. When only a first wash buffer is used (e.g., only the first wash buffer washes and / or suspends the first set of hybridized magnetic particles), the first wash buffer may transport the first set of hybridized magnetic particles 116 to the first detection chamber 120a. In some examples, collecting the first set of hybridized magnetic particles 116 further includes trapping the hybridized plurality of magnetic particles within a threshold distance of the one or more heating elements 122 of the first detection chamber 120.

[0150] Collecting the first set of hybridized magnetic particles may further include transporting the lysate from the waste storage chamber 168 back to the at least one of the one or more extraction chambers 114. Transporting the lysate back to the extraction chamber 114 may occur after transporting the first set of hybridized magnetic particles to the first detection chamberAtty. Dkt. No.: 86357089120a of the one or more detection chambers 120 and before collecting the second set of hybridized magnetic particles. Transporting the lysate back to the extraction chamber 114 may allow a common lysate to be used to hybridize each type of nucleic acids of interest with a corresponding set of magnetic particles having capture oligonucleotides attached that are specific to that type of nucleic acids of interest.

[0151] At process 246, a second set of hybridized magnetic particles 116 are collected. The second set of magnetic particles is hybridized to a second type of nucleic acids of interest. Collecting the second set of hybridized magnetic particles may processes similar to those described above with respect to processes 208-216. At process 246, a second set of hybridized magnetic particles 116 are collected. The second set of hybridized magnetic particles 116 are hybridized to a second type of nucleic acids of interest, using the same lysate used in collection of the first set of hybridized magnetic particles 116. Collecting the second set of hybridized magnetic particles may include processes similar to the processes 208-216 described above.

[0152] For example, collecting the second set of hybridized magnetic particles 116 may include hybridizing, into the at least one of the one or more extraction chambers (e.g., the extraction chamber 114), the second nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles 116 of the plurality of magnetic particles. The at least one capture oligonucleotide may be specific to the second type of nucleic acid of interest. For example, the capture oligonucleotides attached to and / or functionalized with a second set of magnetic particles (e.g., the magnetic particles to be hybridized with the second type of nucleic acids of interest) may have a sequence that is complementary to a sequence of the second type of nucleic acids of interest. This may allow, during hybridization, only the second type of nucleic acids of interest to become hybridized. After the hybridization with the second set of magnetic particles, yet-to-be-targeted nucleotides (e.g., nucleotides not having a sequence complementary to the first or second set of capture oligonucleotides) may remain unhybridized in the extraction chamber 114. The yet-to-be-targeted nucleotides may be hybridized in remaining serial hybridizations.

[0153] Collecting the second set of hybridized magnetic particles 116 may also include holding the second set of hybridized magnetic particles on a side of the extraction chamber 114 (e.g., via an applied magnetic field. Collecting the second set of hybridized magnetic particles 116 may include transporting the lysate to the waste storage chamber 168. Collecting the second set of hybridized magnetic particles 116 may include suspending the second set of hybridizedAtty. Dkt. No.: 86357089magnetic particles in the at least one of the one or more extraction chambers with a second wash buffer. The second wash buffer may be of the same type as the first wash buffer. For example, the cartridge 100 may include a plurality of wash buffer blisters storing the same type of wash buffer. For example, as is shown and described in FIG. 4A, the cartridge of the diagnostic system architecture 400 includes wash buffer blisters 424a and 424b, each of which stores a first type of wash buffer (e.g., the wash buffer 110a). Each set of hybridized magnetic particles that is collected may be washed with the same types of wash buffers (e.g., first wash buffers 110a), but each set of hybridized magnetic particles that is collected is washed and / or suspended with fresh wash buffer (e.g., that is dispensed from separate blister devices) such that no wash buffer is reused.

[0154] Collecting the second set of hybridized magnetic particles 116 may also include transporting the second set of hybridized magnetic particles to a second detection chamber of the one or more detection chambers. For example, the second set of hybridized magnetic particles 116 may be transported to the detection chamber 120b, while the first set of hybridized magnetic particles 116 is transported to the detection chamber 120a. Collecting the second set of hybridized magnetic particles 116 may also include transporting the lysate from the waste storage chamber 168 back to the at least one of the one or more extraction chambers 114 after transporting the second set of hybridized magnetic particles to the second detection chamber 120b of the one or more detection chambers 120. This may allow the lysate to be used to hybridized a third set of magnetic particles with a third set of nucleic acids of interest (e.g., and any further sets of magnetic particles and sets of nucleic acids of interest).

[0155] After collecting the second set of hybridized magnetic particles, the method 200 may include transferring a reconstituted first master mix reagent to the first detection chamber 120 of the one or more detection chambers 120.

[0156] The method 200 may further include delivering a plurality of amplification reagents 125 to the first detection chamber 120a. In various examples, the plurality of amplification reagents 125 may include, for example, a lyophilized master mix reagent, an enzyme for use in amplification, etc. The amplification reagents 125 may be located in various locations of the cartridge (e.g., in blister devices 126) and may be transported to the detection chamber 120 prior to amplification. In various examples, the amplification reagents may be stored as dry or wet reagents. Dry reagents may be dissolved prior to use.Atty. Dkt. No.: 86357089

[0157] Additionally, the method 200 may include transferring a reconstituted second master mix reagent to a second detection chamber 120b of the one or more detection chambers 120 (e.g., when the second set of hybridized magnetic particles are to be delivered to the second detection chamber 120b of the one or more detection chambers 120). The method 200 may further include delivering a plurality of amplification reagents 125 unique to the second set of hybridized magnetic particles to the second detection chamber 120b.

[0158] It should be understood that the method 200 may include collecting any number n sets of hybridized magnetic particles (e.g., 1, 2, 3, 4, 5, etc. number of sets of hybridized magnetic particles). As such the processes described with respect to processes 208-206 may be performed n number of times with n types of nucleic acids of interest, and n sets of magnetic particles attached to capture oligonucleotide specific to a particular type of nucleic acids of interest. Further, the cartridge 100 may include n number of detection chambers, master mix reagents, and amplification reagents such that each type of nucleic acids of interest are delivered to a separate detection chamber and have or use master mix and amplification reagents during amplification that are unique to the specific type of nucleic acid of interest. For example, when 3 serial hybridizations are performed (e.g., 3 sets of hybridized magnetic particles are collected), the cartridge 100 may include first, second, and third detection chambers 120a, 120b, and 120c. First master mix reagents and amplification reagents may be specific to the first type of nucleic acids of interest and may be delivered to the first detection chamber 120a. Similarly, second master mix reagents and amplification reagents may be specific to the second type of nucleic acids of interest and may be delivered to the second detection chamber 120b, and third master mix reagents and amplification reagents may be specific to the third type of nucleic acids of interest and may be delivered to the third detection chamber 120c.

[0159] Referring now to FIG. 3, a method 300 for serial hybridization is shown, according to an example implementation. At process 302, a single lysis is performed. That is, a single lysis is performed on the nucleic acids of interest using an IPC and a system composition / buffer. The resulting lysate is shared for multiple hybridization workflows (e.g., the same lysate is used for multiple serial hybridizations). Use of a single lysate may avoid splitting or dividing the lysate and subsequently losing sensitivity during amplification and detection of the nucleic acids. A single lysate may also avoid use of a large starting sample volume that increases with an increasing number of hybridization workflows.Atty. Dkt. No.: 86357089

[0160] At process 304, hybridization of the lysate to the magnetic particles 116 is performed. Washing of the lysate and delivery of the hybridized magnetic particles 116 is also performed, as described above with respect to FIGS. 2 A and 2B and below with respect to FIGS. 4 A and 4B. Hybridization and washing may be performed in the same chamber (e.g., the extraction chamber 114) such that the lysate does not enter the detection chambers 120.

[0161] At process 306, hybridization, washing, and magnetic particle delivery to the detection chamber 120 (e.g., processes 206-218 of FIG. 2A) are repeated. For example, for n number of samples, an additional n-1 hybridization to magnetic particle delivery workflows are performed. Each sample is delivered to and amplified in a different detection chamber 120. For example, for n number of serial hybridizations performed, the cartridge may include n number of detection chambers 120, and each sample is delivered to a different detection chamber 120. Further, a new or different set of magnetic particles 116 may be used for each sample n. New first and second wash buffers may also be used for each sample n to prevent contamination of samples with magnetic particles used in other samples.

[0162] At process 308, a master mix is reconstituted n number of times. A unique master mix is used for each detection chamber 120. For example, the master mix may have different compositions that are each unique to the sample it is used with. The PCA buffer used in each sample n may be the same, and a specific volume may be metered out for each sample n. The n number of reconstitutions may be performed in parallel and / or during other processes (e.g., hybridization of another sample), to reduce time spent on the overall process.

[0163] At process 310, the PCA amplification reactions for each sample n are performed. The PCA reactions may be performed in parallel. A common heating element 122 may be used to heat all detection chambers 120. Because of the serial hybridization processes, each PCA reaction and corresponding detection may be a low-plex reaction (e.g., detecting below a threshold number of analytes, such as seven analytes). In various implementations, high-Stokes-shift dyes and / or melting curve analysis may be used to increase a number of analytes detected per PCA reaction.

[0164] FIGS.4A and 4B depict an example diagnostic system architectures, in accordance with present implementations. As illustrated by way of example in FIGS. 4A and 4B, an example diagnostic system architecture 400A and 400B, respectively, can include a cartridge and anAtty. Dkt. No.: 86357089instrument. The cartridge may be the same as or similar to the cartridge 100. Further, the instrument may be the same as or similar to the instrument 150.

[0165] The cartridge of the example diagnostic system architecture 400A may include a first vent membrane 404, a plurality of valves 408a-bb, a plurality of frangible seals 410a-l, a liquid sample input chamber 412, a sample filter 414, a p-trap 416, an umbrella valve 418, a plurality of bubble traps 420a-d, a system composition blister device 422, wash buffer blister devices 424a and 424b storing a first wash buffer 110a, wash buffer blister devices 424c and 424d storing a second wash buffer 110b, a fluidic junction 426a, a sample input (SI) metered section 430, an IPC lyo particle 431, a lyse and hybridization chamber 432 (e.g., the extraction chamber 114) having an external ultrasonic horn 434, an infrared (IR) sensor 437, a filter for lysing 439, a plurality of magnetic bead (MB) lyo particles 438a and 438b (e.g., lyophilized particles that are or include magnetic particles 116), a lysate storage chamber 468, a PC A buffer 440 (stored in a blister device), first and second PCA buffer metered sections 442a and 442b, first and second master mix mixing chambers 444a and 444b, each having a master mix lyo bead 446a and 446b (e.g., a lyophilized PC master mix), respectively, a second vent membrane 448, an overflow chamber 454, and first and second PCA chambers 456a and 456b.

[0166] As shown in FIG. 4A, the instrument of the diagnostic system architecture 400A may include a pump 402 (e.g., a syringe pump), a plurality of fluid sensors 428a-g, and first and second external magnets 458a and 458b to interface with a location of the PCA chambers 456a and 456b, respectively, and a third external magnet 458c to interface with a location of the lyse and hybridization chamber 432. The elements of the instrument may be positioned such that certain components interface with certain components of the cartridge. For example, FIG. 4 A indicates locations of fluid sensors 428a-gwithin the instrument relative to the elements within the cartridge. For example, fluid sensors 428a-g may be disposed within the instrument at various locations corresponding to elements located in the cartridge such that the sensors 428a-f sense fluid motion, movement, etc. within the cartridge at the indicated positions. For example, sensor 428a is shown to be located at an inlet of the SI metered section 430. The sensor 428a may not be physically located within the cartridge at the inlet of the SI metered section. Rather, the sensor 428a may be positioned within the instrument such that, upon insertion of the cartridge into the instrument, the position of the sensor 428a aligns with the inlet of the SI metered section 430. Further, as stated, the instrument of the diagnostic system architecture 400 may also include magnets 458a and 458b to interface with the PCA chambersAtty. Dkt. No.: 86357089456a and 456b, respectively, and a magnet 458c to interface with the lyse and hybridization chamber 432. The magnets may be positioned within the instrument such that, upon insertion of the cartridge into the instrument, the location of the magnet 458 aligns with the position of the PCA chamber 456 within the cartridge.

[0167] The system architecture 400B may be similar to the system architecture 400 A. For example, the system architecture 400A may include the components described above. In addition to those components described above, the system architecture 400B may include valves 408cc-hh, frangible seals 410m-p, an additional wash buffer blister 424e storing the first wash buffer 110a, an additional wash buffer blister 424f storing the second wash buffer 110b, an additional MB lyo particle 438c, a third PCA buffer metered section 442c, a third mixing chamber 444c including a third master mix lyo particle 446c, and a third PCA chamber 456c. As shown in FIG. 4B, the instrument of the diagnostic system architecture 400B may include the elements described above with respect to FIG. 4A. In addition, the instrument of the diagnostic system architecture 400B may include fluid sensors 428h-i and a fourth external magnet 458d.

[0168] The system architecture 400A may be implemented in examples in which two serial hybridizations and parallel PCA reactions are performed. The system architecture 400B may be implemented in examples in which three serial hybridizations and parallel PCA reactions are performed. It should be understood that the system architecture may include any number of elements such that any number (e.g., 3, 4, 5, etc.) of serial hybridizations and parallel PCA reactions may be performed. For example, the system architecture may include additional wash buffers 424, valves 408, sensors 428, mixing chambers 444, PCA chambers 456, magnets 458, etc. such that n number of serial hybridizations and PCA reactions can be performed in the cartridge 100.

[0169] In various examples, the system composition blister devices 422 and the wash buffer blister devices 424 may be similar to the blister devices 126a and 126b, respectively. The system composition stored in the blister device 422 may be similar to the system composition and the wash buffer composition stored in the blister devices 424a-424e may be similar to the wash buffer 110. The lyse chamber 432 may be similar to the extraction chamber 114 and the mixing chambers 444 may be similar to the extraction chambers 114. The PCA chambers 456 may also be similar to the detection chamber 120.Atty. Dkt. No.: 86357089

[0170] The following processes and steps may be performed in either of the system architectures 400 A and 400B.

[0171] A user may fill the liquid sample input chamber 412 with a sample liquid. The sample liquid may be a biological sample containing one or more nucleic acids or other molecules of interest. The user may fill the sample input chamber 412 via a pipette, an exact volume pipette, a dropper, syringe injection, etc. The sample input chamber 412 may include fill guides to indicate a fill level. Upon filling the sample input chamber 412, all valves 408 may be open. An umbrella valve 418 may disable the sample input chamber 412 from filling the sample metering circuit (e.g., the SI metered section 430). In various examples, the umbrella valve 418 may be a type of check valve. For example, the umbrella valve 418 may prevent flow back towards the liquid sample input chamber 412. The umbrella valve 418 may have a sufficient cracking pressure in a forward direction, thereby preventing the fluid in the sample input chamber 412 from reaching the sample metering section 430 from gravity (e.g., a head height pressure). Upon filling the liquid sample input chamber 412, the chamber may be closed. For example, a sample input cover may be closed.

[0172] Upon filling the sample input chamber 412, the sample may be pressurized through the valve 408a. Pressure may be vented through normally open valves 408j and / or 408t and the second vent membrane 448. For example, pressure may be vented through valve 408i. Thus, the sample may be pushed through the umbrella valve 418, the bubble trap 420a, and into the sample metering channels (e.g., the SI metered section 430). Fluid flow may be monitored as the liquid moves past an inlet of the SI metered section 430. In various examples, a first fluid flow sensor 428a may be positioned within the instrument such that the sensor aligns with and senses fluid at an inlet of the SI metered section 430. It should be understood that the positions of the fluid flow sensors 428 may be positioned to correspond to any locations of the cartridge 100. For example, a second fluid flow sensor 428b may also be positioned within the instrument such that the sensor aligns with and senses fluid at an outlet of the SI metered section 430. The sensors 428a and 428b may monitor the fluid flow until the liquid reaches a sample metering outlet sensor. Responsive to the liquid reaching the outlet sensor, flow may be stopped. For example, valves may close to prevent movement of the liquid.

[0173] The fluid flow sensors may detect if a cartridge channel or chamber has liquid or air present. The fluid flow sensors may track progress of liquid slugs, meter liquid by triggering the halt of flow to control volume of fluid (e.g., sensors 428b, 428e, 428f and 428g, and 428iAtty. Dkt. No.: 86357089in FIG. 4B), help in reciprocating mixing by triggering when a flow should be reversed (e.g., sensors 428e, and 428f and 428i in FIG. 4B), and / or help locate the reconstituted master mix reagent in the PCA chamber (e.g., sensors 428e, 428f, and 428g, and 428i in FIG. 4B).

[0174] In various examples, sensors from the instrument 150 may be optical and / or capacitive. Optical sensors may be or include LEDs and photodiodes to detect changes in contrast, color, reflection, etc. in a microfluidic channel or chamber. Capacitive sensors may be or include capacitor plates to detect change in dielectric between liquid and air in a microfluidic channel or chamber.

[0175] Responsive to metering the sample, frangible seals 410a and 410b may be opened. Opening the frangible seals 410a and 410b may enable fluid flow into and out of the system composition blister device 422. The cartridge may then be pressurized through the first vent membrane 404 and pinch valve 408b, thus purging the sample from the SI metered section 430. Further, the sample may reconstitute an IPC lyo particle 431 and deliver the IPC lyo particle 431 to the lyse chamber 432, and the sample may fill the lyse chamber 432. Pressure may be vented through one or more of pinch valves 408t and / or 408j and / or the second vent membrane 448 to allow the fluid flow. For example, pressure may be vented through the pinch valve 408j .

[0176] In various examples, the system composition fluid (e.g., system composition ) may flow through the SI metered section 430 as the blister device 422 empties. In various examples, it may be beneficial to fully empty the blister device 422 for improved performance. Air may then be pushed through the blister device 422 (e.g., via syringe pump 402) to fully empty the blister device 422 and purge any remaining system composition fluid from the SI metered section 430 into the lyse chamber 432. Metering sensors (e.g., sensors 428a and 428b) may manage a flow rate through the SI metered section 430 and minimize an amount of air pushed into the lyse chamber 432 from the system composition blister device 422.

[0177] Responsive to emptying the system composition blister device 422, the cartridge 100 may be pressurized through the first vent membrane 404 and the pinch valve 408i. The lyse chamber 432 may then be pressurized to ensure contact between a lyse chamber film and a lyse chamber heater. In various examples, all valves may be closed to isolate the lyse chamber 432 during lysis. In various examples, lysis may be an ultrasonic lysis. As such, the ultrasonic horn 434 may be pulsed, and a temperature of the lyse chamber 432 may be controller via the hornAtty. Dkt. No.: 86357089434, the heater, and / or a temperature sensor. The ultrasonic pulses may lyse the fluid and mix the sample, the system composition, and the IPC lyo particle 431.

[0178] The following description is made in reference to the first MB lyo particle 438a and master mix (MM) mixing chamber 444a.

[0179] Responsive to lysing the sample, the cartridge 100 may be pressurized through the first vent membrane 404 and the pinch valve 408i. . Further the valves 408i, 408m, and / or 408k may be opened. This may allow the lysate (e.g., the sample, the system composition, and the IPC lyo material mixture) to be delivered from the lyse chamber 432 through the location of first MB lyo particle 438a to collect the first MB lyo particle 438a. In some examples, the fluid front may be stopped (e.g., rather than the trailing meniscus) when triggering the sensor 428c before reversing fluid flow. In some examples, the lysate flows into the lysate storage chamber 468. In the lysate storage chamber 468 (or at a location of the first MB lyo particle 438a), a MB lyo particle may be reconstituted. The entrance of the lysate into the lysate storage chamber 468 may cause the first MB lyo particle 438a to be reconstituted. In various examples, all of the lysate is delivered from the lyse chamber 432. Flow may be stopped responsive to a determination that a trailing meniscus of the lysate is detected by the fluid sensor 428c. The lysate storage chamber 468 may be kept full of fluid to minimize air generation in the chamber, which may disrupt amplification. . The valves 408n, 408j, and / or 408k may be actuated to move the lysate back into the lyse chamber 432. This process may be repeated until the MB lyo particle 438a is reconstituted and / or all the lysate has returned to the lyse chamber 432. In some examples, the MB lyo particle 438a is mixed and fully reconstituted using a sonotrode rather than reciprocating flow. The valves 408n, , 408j, and / or 408k may then be closed. In various examples, the sensor 428c may detect when all of the lysate has been purged from the lysate storage chamber 468.

[0180] Responsive to the lysate reentering the lyse chamber 432, hybridization may occur. For example, the lysate may be hybridized. Hybridization may occur due to a chemistry of the lysate, the nucleic acids, the oligonucleotides, and / or the magnetic particles, a temperature of the lyse chamber 432, and / or a duration of the reaction. The ultrasonic horn 434 may be used during hybridization to maintain mixing and add heat to the lyse chamber 432. The magnet 458c may be actuated to trap the magnetic beads in the lyse chamber 432 to a wall or side of the lyse chamber 432. The magnet 458c may interface with the lyse chamber 432 to capture or hold the hybridized magnetic particles to a side or wall of the lyse chamber 432. Trapping theAtty. Dkt. No.: 86357089magnetic particles on the wall of the lyse chamber 432 may allow fluid exchange in and out of the lyse chamber 432 without disposing or removing the magnetic particles 116.

[0181] The valves 408i and 408n may be opened to push the lysate out of the lyse chamber 432 into the lysate storage chamber 468. The lysate moved to the lysate storage chamber 468 may not include the hybridized nucleic acids of interest, capture oligonucleotides, and magnetic particles trapped to the side of the lyse chamber 432.

[0182] In various examples, a plurality of wash buffers may be enabled for use in the cartridge 100. For example, two wash buffers may be used. In various examples, the first wash buffer may be a more aggressive chemistry that may be more inhibitory to a final PCA reaction relative to the second wash buffer. For example, the first wash buffer may include water, 0.017M ofMgCh, 0.1 IM of Tris-HCL for a pH of 8.0, and 0.056% of Tween® 20. In various examples, NaCl or KC1 may be utilized rather than MgCh.

[0183] The first wash buffer may be enabled by opening the frangible seals 410c and / or 410d of the wash buffer blister device 424a. The first wash buffer may then be moved from the blister device 424a to the lyse chamber 432 by pressurizing through the valves 408c and 408g. Valve 408hmay be open to allow fluid flow when negative pressure is used to pull the fluid out from downstream. When positive pressure is used to push the wash buffer out of the wash buffer blister device 424a, valve 408g is opened. The volume of the first wash buffer that is dispensed may based on a predetermined or predefined amount of fluid initially in the wash buffer blister device 424a. In various examples, the blister device 424a may not be fully emptied in order to minimize air passing downstream through a common channel to the lyse chamber 432. After passing a controlled volume of the first wash buffer through the lyse chamber 432, flow may be reversed by pressurizing through valve 408i and opening valve 408x, 408y, and 408c to allow fluid flow. Valve 408j that leads to the vent membrane 448 may then be opened. The first wash buffer may be returned to the blister device 424a until the end of the fluid flow is detected (e.g., by any sensor 428). Responsive to the detection of the end of the fluid flow, additional air and / or liquid may be purged to the lyse chamber 432. The wash buffer 110a may then be returned to the wash buffer blister 424a.

[0184] In some examples, prior to the return of the wash buffer 110a to the blister 424a, the hybridized magnetic particles in the lyse chamber 432 may be resuspended (e.g., via theAtty. Dkt. No.: 86357089ultrasonic horn 434), and the hybridized magnetic particles are again trapped to a side of the lyse chamber 432 using the magnet 458c.

[0185] The second wash buffer 110b may then be transported from the wash buffer blister 424c to the lyse chamber 432. In various examples, the second wash buffer may be a less aggressive chemistry that may be less inhibitory to a final PCA reaction relative to the first wash buffer. For example, the second wash buffer may include 0.01 M Tris buffer composition, 0.1 M NaCl, and 0.0015 M KC1, with a pH 8.0 at 25 degrees Celsius and 0.01% Tween® 20 when dissolved in one liter of deionized water. In some cases, the second wash buffer may include the same or a similar composition to the first wash buffer (e.g., water, 0.017M of MgCh, 0.1 IM of Tris-HCL for a pH of 8.0, and 0.056% of Tween® 20). In some examples, the second wash buffer may be a transport buffer.

[0186] The second wash buffer may be enabled by opening the frangible seals 410g and 41 Oh of the wash buffer blister device 424c. The second wash buffer may then be moved from the blister device 424e to the lyse chamber 432 by pressurizing through the valve 408i. Valves 408x, 408z, and 408e may be open to allow fluid flow. The valve 408j , which leads to the vent membrane 448, may then be opened. The volume of the second wash buffer may be controlled using the sensors 428 positioned within the instrument such that the sensors align with and sense fluid in the lyse chamber 432. In various examples, the blister device 424e may not be fully emptied in order to minimize air passing downstream through a common channel to the lyse chamber 432.

[0187] The magnet 458c may be retracted, causing the hybridized magnetic particles to be released from the side of the lyse chamber 432. Subsequently, the hybridized magnetic particles may be resuspended in the second wash buffer(e.g., via the ultrasonic horn 434).

[0188] The second wash buffer and the suspended hybridized magnetic particles are transported through the PCA chamber 456a. For example, the second wash buffer may be moved from the lyse chamber 432 to the PCA chamber 456a by opening the valves 408t, 408x, 408i, and 408v. The wash buffer and hybridized magnetic particles may be transported through the PCA chamber 456a to capture the magnetic particles onto a heating element (e.g., the heating element 122, a foil, etc.). The magnet 458a may be static and positioned proximate the PCA chamber 456a to capture the magnetic particles. In some examples, the magnet 458a may be actuated to interface with the PCA chamber 456a to capture the magnetic particles.Atty. Dkt. No.: 86357089

[0189] After passing a controlled volume of the second wash buffer through the PCA chamber 456, flow may be reversed by pressurizing through the opened valve 408u and opening valves 408e, 408h, and 408z to allow fluid flow. The second wash buffer may be returned to the blister device 424e until the end of the fluid flow is detected (e.g., by any sensor 428). Responsive to the detection of the end of the fluid flow, additional air and / or liquid may be purged to the lyse chamber 432.

[0190] The master mix lyo bead 446a may be reconstituted and mixed within the master mix mixing chamber 444a. This process may be performed in parallel with the hybridization of the magnetic particles and nucleic acids of interest described above.

[0191] In various examples, the PCA buffer 440 may be metered (e.g., at the PCA buffer metered section 442a). The PCA buffer blister valves 410k and 4101 may be opened. Flow may then move out of the PCA buffer 440. Flow may be moved out by sucking flow through the valve 408q and opening the valve 408o to allow flow. Flow is halted at the sensor 428e. A bubble trap 420d may be used to catch air from the PCA buffer 440 that moves downstream. The PCA buffer 440 may continue to fill the PCA buffer metered section 442a until the PCA buffer 440 is sensed by the PCA buffer metering sensor 428e. To finish metering, valve 408p is opened, valve 408o is closed, and the fluid is pulled into the MM mixing chamber 444a. Mixing may occur by flowing the fluid back and forth between the MM mixing chamber 444a and the PCA buffer metered section 442a. After mixing, valves 408p, 408v, 408u, and 408aa are opened, and the master mix is sucked out of the PCA buffer metered section 442a and into the PCA chamber 456a.

[0192] The PCA buffer 440 may refer to a buffer used to reconstitute a master mix reagent used for reverse transcription and PCA amplification. In some examples PCA buffer 440 comprises water, a salt, a buffer compound (e.g., tris buffer) and optionally a surfactant. In some examples, the salt is MgCh or NaCl. In some examples, the PCA buffer 440 comprises between 0. ImM and 15mM salt. In some examples, the system composition comprises between about 0.001% and about 0.1% (e.g., about 0.001%, 0.01%, 0.05%, or 0.1%) surfactant. In some examples, the surfactant is selected from Tween®20, Tween® 80, Tween® 85, SPAN® 80 or SPAN® 85. In some examples, the surfactant is Tween®-20. In some examples, at least one parameter of the PCA buffer can be adapted to enable hybridization of the target nucleic acid to the functional nucleic acid at a desired complementarity. For example, a concentration of the salt (e.g., MgCh) in the PCA buffer can be increased in order to enable hybridization evenAtty. Dkt. No.: 86357089with low complementarity, whereas optionally the concentration of the salt (e.g., MgCh) in the PCA buffer can be reduced in order to enable hybridization only from a certain higher degree of complementarity.

[0193] After the PCA buffer 440 flows past a location aligned with the sensor 428e, the valve 408o is closed and the valve 408p is opened to allow pressurized air to separate remaining PCA buffer from a controlled metered PCA buffer volume. The metered volume may be sucked into the master mix (MM) mixing chamber 444a, and the master mix lyo bead 446a may be reconstituted. Flow may be stopped responsive to a trailing meniscus of the PCA buffer is sensed by the PCA buffer metering sensor (e.g., the sensor 428e).

[0194] The master mix (MM) lyo material may be mixed well into the PCA buffer. The MM lyo material may be mixed into the PCA buffer by recirculating flow between the MM metering section and the MM mixing chamber 444. Flow may be recirculated by alternating positive and negative pump pressure with valve 408q and valve 408p open. Once mixing is complete, the master mix is pulled into the metering channels and out of the mixing chamber 444.

[0195] Responsive to the master mix being fully mixed, the master mix may be loaded into the PCA chamber 456a. The master mix may enter the PCA chamber 456a by applying a vacuum to valves 408v and 408u, with valves 408aa and 408p open to allow fluid flow. The master mix may enter the PCA well until a trailing liquid meniscus of the master mix arrives at a location corresponding to the PCA inlet sensor 428g.

[0196] The steps described above (e.g., beginning with transporting the lysate to the lysate storage chamber 468) may be repeated for the second MB lyo particle 438b and master mix lyo particle 446b and / or for the third MB lyo particle 438c and master mix lyo particle 446c (e.g., in the example described with respect to FIG. 4B). These steps may be repeated n number of times for n number of hybridizations / MB lyo parti cles / master mix lyo particles.

[0197] Below is a description of the processes described above for the second MB lyo particle 438b and MM lyo particle 446b.

[0198] Responsive to lysing the sample, the cartridge 100 may be pressurized through the first vent membrane 404 and the pinch valve 408i. Further, the valves 408i, 4081, and / or 408mmay be opened. This may allow the lysate (e.g., the sample, the system composition, and the IPC lyo material mixture) to be delivered from the lyse chamber 432 through the location of theAtty. Dkt. No.: 86357089second MB lyo particle 438b and, optionally, into the lysate storage chamber 468. In some examples, the fluid front is stopped (e.g., rather than a trailing meniscus) when triggering the sensor 428d prior to reversing fluid flow. In the lysate storage chamber 468 or at the location of the second MB lyo particle 438b, the MB lyo particle may be reconstituted. The entrance of the lysate into the lysate storage chamber 468 may cause the second MB lyo particle 438b to be reconstituted. In various examples, all of the lysate is delivered from the lyse chamber 432. Flow may be stopped responsive to a determination that a trailing meniscus of the lysate is detected by the fluid sensor 428d. . The lysate storage chamber 468 may be kept full of fluid to minimize air generation in the chamber, which may disrupt amplification. The valves 408n, 4081, and / or 408j may be actuated to move the lysate back into the lyse chamber 432. This process may be repeated until the MB lyo particle 438b is reconstituted and / or all the lysate has returned to the lyse chamber 432. In some examples, the MB lyo particle 438b is mixed and fully reconstituted using a sonotrode rather than reciprocating flow. The valves 408n, 4081, and / or 408j may then be closed. In various examples, the sensor 428c may detect when all of the lysate has been purged from the lysate storage chamber 468.

[0199] Responsive to the lysate reentering the lyse chamber 432, hybridization may occur. For example, the lysate may be hybridized. Hybridization may occur due to a chemistry of the lysate, the nucleic acids, the oligonucleotides, and / or the magnetic particles, a temperature of the lyse chamber 432, and / or a duration of the reaction. The ultrasonic horn 434 may be used during hybridization to maintain mixing and add heat to the lyse chamber 432.. The magnet 458c may be actuated to trap the magnetic beads in the lyse chamber 432 to a wall or side of the lyse chamber 432. The magnet 458c may interface with the lyse chamber 432 to capture or hold the hybridized magnetic particles to a side or wall of the lyse chamber 432. Trapping the magnetic particles on the wall of the lyse chamber 432 may allow fluid exchange in and out of the lyse chamber 432 without disposing or removing the magnetic particles 116.

[0200] The valves 408i, 4081 and / or 408n may be opened to push the lysate out of the lyse chamber 432 into the lysate storage chamber 468. Valve 408x may remain closed when lysate is being pushed out of the lyse chamber 432 to precent lysate from reaching the PCA chambers 456. The lysate moved to the lysate storage chamber 468 may not include the hybridized nucleic acids of interest, capture oligonucleotides, and magnetic particles trapped to the side of the lyse chamber 432.Atty. Dkt. No.: 86357089

[0201] The first wash buffer may be enabled by opening the frangible seals 410e and / or 41 Of of the wash buffer blister device 424b. The first wash buffer may then be moved from the blister device 424b to the lyse chamber 432 by pressurizing through the valves 408d and 408g. Valve 408x may be open to allow fluid flow. Valve 408g may also be opened when positive pressure is used to push the wash buffer out of the wash buffer blister device 424b. Valve 408h may be opened instead of valve 408g when negative pressure is used to pull the wash buffer out from downstream. The volume of the first wash buffer that is dispensed may based on a predetermined or predefined amount of fluid initially in the wash buffer blister device 424a In various examples, the blister device 424b may not be fully emptied in order to minimize air passing downstream through a common channel to the lyse chamber 432. After passing a controlled volume of the first wash buffer through the lyse chamber 432, flow may be reversed by pressurizing valve 408i and opening valves 408x, 408y, and 408d to allow fluid flow. The first wash buffer may be returned to the blister device 424b until the end of the fluid flow is detected (e.g., by any sensor 428). Responsive to the detection of the end of the fluid flow, additional air and / or liquid may be purged to the lyse chamber 432. The wash buffer 110a may then be returned to the wash buffer blister 424b.

[0202] In some examples, prior to the return of the wash buffer 110a to the blister 424b, the hybridized magnetic particles in the lyse chamber 432 may be resuspended (e.g., via the ultrasonic horn 434), and the hybridized magnetic particles are again trapped to a side of the lyse chamber 432 using the magnet 458c.

[0203] The second wash buffer 110b may then be transported from the wash buffer blister 424d to the lyse chamber 432. The second wash buffer may be enabled by opening the frangible seals 41 Oi and 41 Oj of the wash buffer blister device 424d. The second wash buffer may then be moved from the blister device 424d to the lyse chamber 432 by pressurizing through the valve 408i. Valves 408x, 408z, and 408f may be open to allow fluid flow. The volume of the second wash buffer may be controlled using the sensors 428 positioned within the instrument such that the sensors align with and sense fluid in the lyse chamber 432. In various examples, the blister device 424d may not be fully emptied in order to minimize air passing downstream through a common channel to the lyse chamber 432.

[0204] The magnet 458c may be retracted, causing the hybridized magnetic particles to be released from the side of the lyse chamber 432. Subsequently, the hybridized magnetic particles may be resuspended in the second wash buffer (e.g., via the ultrasonic horn 434).Atty. Dkt. No.: 86357089

[0205] The second wash buffer and the suspended hybridized magnetic particles are transported through the PCA chamber 456b. For example, the second wash buffer may be moved from the lyse chamber 432 to the PCA chamber 456b by opening the valves 408t, 408x, 408i, , and 408w The wash buffer and hybridized magnetic particles may be transported through the PCA chamber 456b to capture the magnetic particles onto a heating element (e.g., the heating element 122, a foil, etc.). The magnet 458b may be static and positioned proximate the PCA chamber 456b to capture the magnetic particles. In some examples, the magnet 458b may be actuated to interface with the PCA chamber 456b to capture the magnetic particles.

[0206] After passing a controlled volume of the second wash buffer through the PCA chamber 456, flow may be reversed by pressurizing through the opened valve 408u and opening valves 408f, 408h, and 408z to allow fluid flow. The second wash buffer may be returned to the blister device 424d until the end of the fluid flow is detected (e.g., by any sensor 428). Responsive to the detection of the end of the fluid flow, additional air and / or liquid may be purged to the lyse chamber 432.

[0207] The master mix lyo particle 446b may be reconstituted and mixed within the master mix mixing chamber 444b. This process may be performed in parallel with the hybridization of the magnetic particles and nucleic acids of interest described above.

[0208] In various examples, the PCA buffer 440 may be metered (e.g., at the PCA buffer metered section 442b). The PCA buffer blister valves 410k and 4101 may be opened. Flow may then move out of the PCA buffer 440. Flow may be moved out by sucking flow at the valve 408s and opening the valve408o to allow flow. Flow is halted at a location corresponding to sensor 428f. A bubble trap 420d may be used to catch air from the PCA buffer 440 that moves downstream. The PCA buffer 440 may continue to fill the PCA buffer metered section 442b until the PCA buffer 440 is sensed by the PCA buffer metering sensor 428f. To finish metering, valve 408r is opened, valve 408o is closed, and the fluid is pulled into the MM mixing chamber 444b. Mixing may occur by flowing the fluid back and forth between the MM mixing chamber 444b and the PCA buffer metered section 442b. After mixing, valves 408r, 408w, 408u, and 408bb are opened, and the master mix is sucked out of the PCA buffer metered section 442a and into the PCA chamber 456b.

[0209] After the PCA buffer flows past a location aligned with the sensor 428f, the valve 408o is closed and the valve 408r is opened to allow pressurized air to separate remaining PCA bufferAtty. Dkt. No.: 86357089from a controlled metered PCA buffer volume. The metered volume may be sucked into the master mix (MM) mixing chamber 444b, and the master mix lyo particle 446b may be reconstituted. Flow may be stopped responsive to a trailing meniscus of the PCA buffer is sensed by the PCA buffer metering sensor (e.g., the sensor 428f).

[0210] The master mix (MM) lyo material may be mixed well into the PCA buffer. The MM lyo material may be mixed into the PCA buffer by recirculating flow between the MM metering section and the MM mixing chamber 444. Flow may be recirculated by alternating positive and negative pump pressure with valve 408s and valve 408r open. Once mixing is complete, the master mix is pulled into the metering channels and out of the mixing chamber 444.

[0211] Responsive to the master mix being fully mixed, the master mix may be loaded into the PCA chamber 456b. The master mix may enter the PCA chamber 456b by applying a vacuum to valve 408w and 408u, with valves 408bb and 408r open to allow fluid flow. The master mix may enter the PCA well until a trailing liquid meniscus of the master mix arrives at a location corresponding to the PCA inlet sensor 428g.

[0212] Below is a description of the processes described above for the third MB lyo particle 438c and MM lyo particle 446c.

[0213] Responsive to lysing the sample, the cartridge 100 may be pressurized through the first vent membrane 404 and the pinch valve 408i. Further the valves 408i, 408m, and / or 408ee may be opened. This may allow the lysate (e.g., the sample, the system composition, and the IPC lyo material mixture) to be delivered from the lyse chamber 432 through the location of third MB lyo particle 438c to collect the third MB lyo particle 438c. In some examples, the fluid front may be stopped (e.g., rather than the trailing meniscus) when triggering the sensor 428h before reversing fluid flow. In some examples, the lysate flows into the lysate storage chamber 468. In the lysate storage chamber 468 (or at a location of the third MB lyo particle 438c) the MB lyo particle 438c may be reconstituted. The entrance of the lysate into the lysate storage chamber 468 may cause the third MB lyo particle 438c to be reconstituted. In various examples, all of the lysate is delivered from the lyse chamber 432. Flow may be stopped responsive to a determination that a trailing meniscus of the lysate is detected by the fluid sensor 428c. The lysate storage chamber 468 may be kept full of fluid to minimize air generation in the chamber, which may disrupt amplification. The valves 408n, 408j, and / or 408ee may be actuated to move the lysate back into the lyse chamber 432. This process may be repeated until the MB lyoAtty. Dkt. No.: 86357089particle 438c is reconstituted and / or all the lysate has returned to the lyse chamber 432. In some examples, the MB lyo particle 438c may be mixed and fully reconstituted using the sonotrode rather than reciprocating flow. The valves 408n, 408j, and / or 408ee may then be closed. In various examples, the sensor 428c may detect when all of the lysate has been purged from the lysate storage chamber 468.

[0214] Responsive to the lysate reentering the lyse chamber 432, hybridization may occur. Hybridization may occur due to a chemistry of the lysate, the nucleic acids, the oligonucleotides, and / or the magnetic particles, a temperature of the lyse chamber 432, and / or a duration of the reaction. The ultrasonic horn 434 may be used during hybridization to maintain mixing and add heat to the lyse chamber 432. The magnet 458c may be actuated to capture or hold the hybridized magnetic particles to a side or wall of the lyse chamber 432. Trapping the magnetic particles on the wall of the lyse chamber 432 may allow fluid exchange in and out of the lyse chamber 432 without disposing or removing the magnetic particles 116.

[0215] The valves 408i, 408n, and / or 408ee may be opened to push the lysate out of the lyse chamber 432 into the lysate storage chamber 468. In various examples, valve 408x may be closed when lysate is being transported out of the lyse chamber 432 to prevent lysate from reaching the PCA chambers 456. Valve 408o may be open only when wash buffer is being transported. The lysate moved to the lysate storage chamber 468 may not include the hybridized nucleic acids of interest, capture oligonucleotides, and magnetic particles trapped to the side of the lyse chamber 432.

[0216] The first wash buffer may be enabled by opening the frangible seals 410m and / or 41 On of the wash buffer blister device 424e. The first wash buffer may then be moved from the blister device 424e to the lyse chamber 432 by pressurizing through the valves 408d and 408g. Valve 408g may be open to allow fluid flow when positive pressure is used to push the wash buffer out of the blister device 424e. Valve 408h may be open when negative pressure is used to pull the wash buffer out from downstream. The volume of the first wash buffer that is dispensed may based on a predetermined or predefined amount of fluid initially in the wash buffer blister device 424e. In various examples, the blister device 424e may not be fully emptied in order to minimize air passing downstream through a common channel to the lyse chamber 432. After passing a controlled volume of the first wash buffer through the lyse chamber 432, flow may be reversed by pressurizing valve 408i and opening valves 408x, 408y, and / or 408cc to allow fluid flow. Valve 408h may then be opened, which leads to the vent membrane 448. The firstAtty. Dkt. No.: 86357089wash buffer may be returned to the blister device 424e until the end of the fluid flow is detected (e.g., by any sensor 428). Responsive to the detection of the end of the fluid flow, additional air and / or liquid may be purged to the lyse chamber 432. The wash buffer 110a may then be returned to the wash buffer blister 424e.

[0217] In some examples, prior to the return of the wash buffer 110a to the blister 424e, the hybridized magnetic particles in the lyse chamber 432 may be resuspended (e.g., via the ultrasonic horn 434), and the hybridized magnetic particles are again trapped to a side of the lyse chamber 432 using the magnet 458c.

[0218] The second wash buffer 110b may then be transported from the wash buffer blister 424f to the lyse chamber 432. The second wash buffer may be enabled by opening the frangible seals 410o and 41 Op of the wash buffer blister device 424f. The second wash buffer may then be moved from the blister device 424f to the lyse chamber 432 by pressurizing through the valve 408i. Valves 408x, 408z, and / or 408dd may be open to allow fluid flow. The volume of the second wash buffer may be controlled using the sensors 428 positioned within the instrument such that the sensors align with and sense fluid in the lyse chamber 432. In various examples, the blister device 424f may not be fully emptied in order to minimize air passing downstream through a common channel to the lyse chamber 432.

[0219] The magnet 458c may be retracted, causing the hybridized magnetic particles to be released from the side of the lyse chamber 432. Subsequently, the hybridized magnetic particles may be resuspended in the second wash buffer (e.g., via the ultrasonic horn 434).

[0220] The second wash buffer and the suspended hybridized magnetic particles are transported through the PCA chamber 456c. For example, the second wash buffer may be moved from the lyse chamber 432 to the PCA chamber 456c by opening the valves 408t, 408x, 408i, and 408hh. The wash buffer and hybridized magnetic particles may be transported through the PCA chamber 456c to capture the magnetic particles onto a heating element (e.g., the heating element 122, a foil, etc.). The magnet 458d may be static and positioned proximate the PCA chamber 456c to capture the magnetic particles. In some examples, the magnet 458d may be actuated to interface with the PCA chamber 456c to capture the magnetic particles.

[0221] After passing a controlled volume of the second wash buffer through the PCA chamber 456, flow may be reversed by pressurizing through the opened valve 408u and opening valves 408dd, 408h, and 408z to allow fluid flow. The second wash buffer may be returned to theAtty. Dkt. No.: 86357089blister device 424f until the end of the fluid flow is detected (e.g., by any sensor 428). Responsive to the detection of the end of the fluid flow, additional air and / or liquid may be purged to the lyse chamber 432.

[0222] The master mix lyo particle 446c may be reconstituted and mixed within the master mix mixing chamber 444c. This process may be performed in parallel with the hybridization of the magnetic particles and nucleic acids of interest described above.

[0223] In various examples, the PCA buffer 440 may be metered (e.g., at the PCA buffer metered section 442c). The PCA buffer blister valves 410k and 4101 may be opened. Flow may then move out of the PCA buffer 440. Flow may be moved out by sucking flow at the valve 408gg and opening the valve 408o to allow flow. Flow may be halted at a position corresponding to a location of the sensor 428i. A bubble trap 420d may be used to catch air from the PCA buffer 440 that moves downstream. The PCA buffer 440 may continue to fill the PCA buffer metered section 442c until the PCA buffer 440 is sensed by the PCA buffer metering sensor 428i. To finish metering, valve 408ff is opened, valve 408o is closed, and the fluid is pulled into the MM mixing chamber 444c. Mixing may occur by flowing the fluid back and forth between the MM mixing chamber 444c and the PCA buffer metered section 442c. After mixing, valves 408ff, 408hh, 408u, and 408cc are opened, and the master mix is sucked out of the PCA buffer metered section 442c and into the PCA chamber 456c.

[0224] After the PCA buffer flows past a location aligned with the sensor 428f, the valve 408o is closed and the valve 408ff is opened to allow pressurized air to separate remaining PCA buffer from a controlled metered PCA buffer volume. The metered volume may be sucked into the master mix (MM) mixing chamber 444c, and the master mix lyo particle 446c may be reconstituted. Flow may be stopped responsive to a trailing meniscus of the PCA buffer is sensed by the PCA buffer metering sensor (e.g., the sensor 428f).

[0225] The master mix (MM) lyo material may be mixed well into the PCA buffer. The MM lyo material may be mixed into the PCA buffer by recirculating flow between the MM metering section and the MM mixing chamber 444c. Flow may be recirculated by alternating positive and negative pump pressure with valve 408gg and valve 408ff open. Once mixing is complete, the master mix is pulled into the metering channels and out of the mixing chamber 444c.

[0226] Responsive to the master mix being fully mixed, the master mix may be loaded into the PCA chamber 456c. The master mix may enter the PCA chamber 456c by applying a vacuumAtty. Dkt. No.: 86357089to valves 408u and 408hh, with valves 408cc and 408ff open to allow fluid flow. The master mix may enter the PCA well until a trailing liquid meniscus of the master mix arrives at a location corresponding to the PCA inlet sensor 428g.

[0227] Responsive to the master mix arriving at each of the PCA chambers 456, PCA may be performed in each of the chambers. The PCA reactions may be performed in parallel. The PCA chamber 456 may be pressurized via the valve 408u to reduce an impact of air bubble growth during PCA temperature cycling. After pressurization, the chamber may be isolated from the pump by closing all valves of the cartridge 100. An isothermal temperature in the PCA chambers 456 may be controlled via a plurality of heaters on each side of the PCA chambers 456. In various examples, electrical pulsing of the PCA foil (e.g., the heating element 122) may create temperature pulses for the PCA reactions.

[0228] Referring now to FIG. 5A, a cross-sectional view of the detection chamber 120 is shown, according to an example implementation. The detection chamber 120 may include a plurality of temperature regulators 502 surrounding the detection chamber 120, a plastic layer 506, an adhesive 508, and a heat spreader 510. In various examples, a detection chamber well 504 may be a cavity formed between the plastic layer 506 and the heating element 122. The detection chamber well 504 may be configured to house the nucleic acids of interest for amplification and detection. The heating element 122 may be coupled to an energy source, which is coupled to a controller and the electrical components. In various examples, the electrical components, the controller, and / or the energy source may be located in the instrument 150. In various examples, the detection chamber 120 may also be referred to as an amplification chamber, a reaction chamber, a PCA chamber, a PCA reaction chamber, etc. In various examples, the cartridge 100 may include a plurality of detection chambers and / or other types of chambers. For example, the cartridge 100 may include an amplification chamber and a detection chamber.

[0229] The detection chamber 120 may be configured to house an amplification reaction. Amplification may be performed using a nucleic acid amplification method selected from one or more of: pulse-controlled amplification (PCA), reverse transcriptase pulse controlled amplification (RT-PCA), polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR) or real-time polymerase chain reaction (qPCR). In some examples, the step of amplifying the nucleic acid of interest is done by pulse-controlled amplification (PCA).Atty. Dkt. No.: 86357089

[0230] PCA reactions may be utilized to amplify the molecule of interest. Specifically, in said PCA reactions, only a portion of the chamber in which the reaction occurs may be heated for amplification, as opposed to the entirety of the reaction chamber. This may facilitate optimization of temperature control and provide for a more efficient amplification. Compared to polymerase chain reactions (PCR) for amplification, PCA reaction cycles may have a decreased duration, thus decreasing an amount of time for amplification and detection to occur.

[0231] In various examples, during the PCA reaction, a small volume of the fluid in the detection chamber well 504 may be temporarily heated (e.g., thermocycled). For example, 99% of the fluid volume may be unheated, and 1% of the fluid volume may be temporarily heated during the PCA reaction. For example, 99% of the fluid volume may remain at an isothermal temperature (e.g., between 60 and 70 degrees Celsius), while the 1% of the fluid volume being heated may temporarily heat to between 90 and 110 degrees Celsius. For example, the isothermal volume may remain at 65 degrees Celsius, while the heated fluid volume may be temporarily heated to 100 degrees Celsius (and return to 65 degrees Celsius when electrical pulses are not being delivered to temporarily heat the heating element 122).

[0232] As shown in FIG. 5 A, line 511 indicates a border of a denaturation zone. For example, under the line 511, the magnetic particles 116 are shown. During the PCA reaction, nucleic acids of interest may be attached to the magnetic particles 116 and may generally stay within the area outlined by the line 511. Thus, in various examples, the denaturation zone may be a volume of the detection chamber well 504 (e.g., about 15 micrometer thick) where the detection chamber 120 is locally heated to perform the PCA reaction. As described herein, denaturation may describe the separation of a nucleic acid into its two single strands. Denaturation may allow for amplification of the nucleic acids of interest, as each single strand may be used to replicate another strand, increasing the number of nucleic acid strands. Further, the denaturation zone of the detection chamber 120 may be the portion of the chamber that is heated, while the temperature of the remainder of the detection chamber 120 is unchanged. For example, the heating element 122 may deliver pulses and generate heat so that the temperature of the denaturation zone increases. As described herein, the pulses may be configured such that the generated heat dissipates quickly and does not cause a temperature change to the detection chamber well 504 as a whole (e.g., the overall chamber is isothermal but the denaturation zone experiences a temperature increase).Atty. Dkt. No.: 86357089

[0233] In some examples, more than one nucleic acid of interest may be amplified by thermocycling. Thermocycling may be performed when the magnetic particles 116 are functionalized with different capture oligonucleotides designed to be linked to different nucleic acid of interest.

[0234] The temperature regulators 502 may be or include heatsinks. The temperature regulators 502 may be heating and / or cooling elements configured to bring a temperature of the system to an isothermal temperature above room temperature. For example, the temperature regulators 502 may raise an isothermal temperature of the detection chamber 120 to between 60 and 70 degrees Celsius. For example, the isothermal temperature of the detection chamber may be raised to 65 degrees Celsius. As the heating element 122 is delivered an electrical pulse to locally heat a portion of the detection chamber 120, the temperature regulators 502 may remove heat from the detection chamber 120 to prevent a temperature of the overall detection chamber 120 from raising beyond the isothermal temperature. In various examples, a temperature sensor may be located on or proximate the detection chamber 120. The temperature sensor may monitor the temperature of the detection chamber in various locations. For example, a temperature sensor may monitor a temperature at or near the heating element 122 and / or a temperature away from the heating element 122 (e.g., a location at which the temperature should remain constant or relatively constant). The temperature data may be sent to a controller. Responsive to receiving the temperature data, the controller may activate or otherwise control the pulses delivered by the energy source.

[0235] The plastic layer 506 may be a first layer of the detection chamber 120. In various examples the plastic layer 506 may be a few hundred micrometers thick (e.g., around 200 micrometers thick). The plastic may be or include a base of the cartridge 100. For example, the plastic layer 506 may be the same material that the base of the cartridge 100 is made out of. For example, the plastic layer 506 may also be referred to as the cartridge base layer 506. The cartridge may be made of a dielectric material. In some examples, the walls of the cartridge may comprise a polymer material, such as (but not limited to) a cyclic olefin copolymer (COC) material. In some examples, the polymer material may comprise polyethylene, polypropylene, polycarbonate, polymethylmethacrylate (PMMA), and the like.

[0236] The heating element 122 may be coupled to or adjacent to the denaturation zone (e.g., marked by the line 511). The heating element 122 may be a resistive local heating element. Particularly, the heating element 122 may be a foil (e.g., a metal foil). Heating of the heatingAtty. Dkt. No.: 86357089element 122 may be achieved by means of short electrical pulses with which the local heating element(s) 122 are energized. For example, the energy source may be coupled to the heating element 122. The controller may control the energy source to generate pulses to the heating element 122 to heat the heating element 122, and, subsequently, a portion of the detection chamber well 504.

[0237] In some examples, generating pulses of energy may be performed such that only the immediate vicinity of the heating element 122 is heated locally for a short time. Heating of the heating element 122 may allow denaturation of the nucleic acid molecules in the reaction volume, while the bulk of the reaction volume (i.e., the reaction solution) may remain at a base temperature at which elongation and / or hybridization can take place.

[0238] When a current flows through the heating element 122 the heating element 122 may begin to heat up at the beginning of the heating pulse.

[0239] In various examples, the heating element 122 may be around ten micrometers thick. To realize the lowest possible heat capacity, the heating element 122 may have a thickness of less than 100 micrometers in at least one dimension (e.g., less than 50 micrometers, less than 30 micrometers). The thickness of the heating element 122 may be sufficiently low to provide sufficient electrical resistance or impedance. For example, a thinner heating element 122 may have an increased resistance and, consequently, allow for greater Joule heating. In order to make the heating element 122 not too fragile, the material thickness in each dimension may be at least 100 nm, at least 1 micrometers and / or 5 micrometers or 10 micrometers. In some examples, the heating element 122 may comprise a thickness of about 10 to about 50 microns, a thickness of about 15 to about 40 microns, or a thickness of about 20 to about 30 microns, and in some examples, a thickness of about 25 microns.

[0240] In some examples, the heating element 122 may be formed of a metallic foil. For example, the heating element 122 may be formed of ferromagnetic materials such as steel, stainless steels, nickel, and / or highly conductive non-ferrous metals, such as brass and / or copper. In some examples, the heating element 122 may comprise a material such as: stainless steel, brass, titanium, tantalum, tungsten, aluminum, copper, platinum, gold, silver, zinc, indium tin oxide (ITO), and combinations thereof. In some such examples, the first layer material is a stainless-steel material. Alternatively or additionally, the heating element 122 may be at least partially formed of very hard materials, such as tungsten, which may allow very thinAtty. Dkt. No.: 86357089designs of the local heating element 122. In addition, the heating element 122 may have a very high thermal conductivity.

[0241] In some examples, the heating element 122 forms at least a part of a container wall of the reaction container. This may allow for direct contact between the heating element 122 and the sample fluid or reaction solution to be established in a simple manner.

[0242] In various examples, the heating element 122 may be a first layer of a plurality of layers comprising the detection chamber 120. The first layer (e.g., the heating element 122, also referred to herein as a “heating element” or a “foil”) may comprise an electrically activatable heating element that may be to generate heat within the detection chamber 120. In some examples, the heating element 122 may comprise an electrically conductive material. Upon application of an electrical signal to induce heating, the electrically conductive material (e.g., the heating element 122) may generate power (P) depending on its resistivity (R) and current (I) where P=I2x R. Accordingly, in some instances, the heating element 122 also may sometimes be referred to as being an electrically resistive sheet. In various examples, the heating element 122 may be aPCA foil. For example, the heating element 122 may be stainless steel.

[0243] In various examples, the foil (e.g., the heating element 122 or the first layer) may be wider than the PCA or detection chamber 120. This may allow electrical probes of the instrument 150 (e.g., the energy source) to contact the detection chamber 120, specifically the heating element 122. This may allow for the generation of the electrical pulses for PCA.

[0244] In some examples, a wall of the detection chamber 120 may further comprise a second sheet or layer. The second sheet may be the adhesive 508 or a different second layer. The second sheet may act to electrically isolate the first layer from a third layer (e.g., heat spreader 510. In some examples, the adhesive 508 may comprise a thickness of about 10 microns to about 200 microns. In some examples, the adhesive 508 comprises an adhesive layer, such as a pressure sensitive adhesive (PSA) layer. In some examples the adhesive layer 508 may comprise a PSA layer having a thickness up to 200 microns. In some examples, the adhesive layer 508 may be a heat spreader pressure sensitive adhesive. In some examples, the adhesive layer 508 may comprise a material including both thermosetting and thermoplastic properties. In some such examples, the material of the adhesive layer 508 may comprise acrylic adhesive materials. In some of these examples, the adhesive layer 508 may comprise a thermal bondingAtty. Dkt. No.: 86357089adhesive, such as but not limited to: a Pyralux®-based material from DuPont de Nemours, Inc. of Wilmington, Delaware; and a FastelFilm material obtainable from Fastel Adhesives and Substrate Products via www.fasteladhesives.com; and the like.

[0245] In some examples, the wall of the detection chamber comprises a third layer. The third layer may be a heat spreader 510. The heat spreader 510 may be located in the detection chamber 120. In various examples, the heat spreader 510 may directly contact the cartridgecontact heater in the instrument 150. For example, the cartridge-contact heater may interface with the heat spreader 510 to heat each of the one or more detection chambers. That is, the heat spreader 510 may be or include a thermally conductive elastomer plate or a metal plate and may be to transfer heat from the cartridge-contact heater to the detection chamber 120. Specifically, the heat spreader 510 can be or include one or more of a thermally conductive elastomer plate or a metal plate operable as a thermal conduit for transferring heat between the cartridge-contact heater and the detection chamber 120.

[0246] In some examples, the heat spreader 510 comprises a thermally conductive metal sheet. In some examples, the heat spreader 510 may comprise a material such as: aluminum, copper, brass, and combinations thereof or other thermally conductive materials. In some other examples, the heat spreader 510 comprises aluminum. A metal heat spreader 510 may, due to its rigidity, provide worse thermal contact relative to an elastomer. However, a metal heat spreader 510 may rapidly spread heat flow laterally throughout the portion of the detection chamber 120 that is heated during PCA.

[0247] In various examples, the heat spreader 510 may be an elastomer. For example, the heat spreader 510 may be a fiberglass reinforced silicone film. Specifically, the heat spreader 510 may be a thermally conductive elastomer. An elastomer heat spreader 510 may provide better thermal contact compared to a metal heat spreader due to a lower thermal contact impedance, but may not actually “spread” heat through the detection chamber 120.

[0248] The heat spreader 510 may comprise a thickness of about 150 microns to about 500 microns, and in some examples a thickness of about 250 to about 400 microns. The thickness provides a mechanical stiffness sufficient to resist or prevent deformation of the heating element 122.Atty. Dkt. No.: 86357089

[0249] In various examples, the adhesive 508 and / or the heat spreader 510 may be optional. Thus, in various examples, the one or more heating elements of the detection chamber 120 includes only the heating element 122 (e.g., a foil).

[0250] Referring now to FIG. 5B, a system 500B including the detection chamber 120 is shown, according to an example implementation. The system 500B may be the same as or similar to 500A. Further, components of the system 500B may be the same as or similar to the components of the system 500A. For example, the system 500B shows a detection chamber 120 having the detection chamber well 504, the cartridge base (or plastic layer) 506, the heating element 122, the adhesive 508, and the heat spreader 510. In various examples, the system 500B may include a second adhesive 512.

[0251] For example, in various examples, the second adhesive 512 may be a foil PSA layer. The foil PSA layer may be a three layer element. For example, the foil PSA layer may include an adhesive layer, a backer layer, and another adhesive layer.

[0252] The system 500B of FIG. 5B further shows an optical film 514 attached to, coupled to, or otherwise affixed to the detection chamber 120. In various examples, the optical film 514 may allow detection of the amplified nucleic acids of interest. For example, the optical film 514 may have high transparency and / or low haze. This may allow the optical unit 162 of the instrument 150 to detect a presence, absence, and / or amount of a nucleic acid of interest. For example, during the PCA reaction, the nucleic acids may be tagged with a fluorophore. The optical unit 162 may utilize, for example, a sensor within the detection chamber 120 to detect fluorescence corresponding to an amount of the nucleic acid of interest.

[0253] Referring now to FIG. 6, a method 600 for detecting the presence, absence, amount, etc. of a nucleic acid of interest, according to some examples. Generally, a user may input an amount of sample (e.g., such as swab specimen eluted in a transport media) into the cartridge 100. The cartridge 100 may meter a proper amount of the sample and system composition, and internal positive control (IPC) may be added. The target organisms in the sample and IPC are lysed. Multiple samples may then be prepared sequentially. Specifically, nucleic acids from target organisms and IPC may hybridize (e.g., bind) to paramagnetic particles. Paramagnetic particles with bound capture oligonucleotides may be washed with a wash buffer 110 and captured onto a pulse heater in the detection chamber 120 with a magnetic field. PCA buffer may be metered and used to reconstitute Master Mix reagent used for PCA. ReconstitutedAtty. Dkt. No.: 86357089Master Mix reagent may be loaded into the detection chamber 120 with trapped magnetic particles. Sample preparation may occur for any n number of samples. PCA (e.g., RT-PCA) is performed on each sample with real-time multi-channel detection. Results of PCA and detection of target nucleic acids are reported to the user.

[0254] At block 602, a specimen sample may be collected. The specimen sample may be a biological sample collected from, for example, a human that contains a molecule of interest. For example, the biological sample may include one or more nucleic acids of interest. A molecule of interest may be a molecule to be amplified and detected. The molecule of interest may be used for various purposes, such as diagnosing the person that the specimen sample belongs to. In various examples, the specimen sample may be collected by a nasal swab or other retrieval device. Further, the specimen sample may be collected from the person and eluted in a commercial transport medium, such as Copan UTM ®, to be input into the cartridge 100. At block 604, the sample may be input into the cartridge 100. For example, the sample may be input into a sample input chamber. The sample volume of the specimen may be greater than a predefined value (e.g., between 275 and 325 microliters). For example, the sample volume may be greater than 300 microliters. At block 606, the volume of the specimen inserted into the cartridge may be metered so that the sample volume used in the detection process is at or around the predefined value. For example, 320 microliters of the specimen may be collected, and the volume may be metered to 300 microliters to be inserted into the cartridge. Metering a sample may include, for example, utilizing the sample as stored in the cartridge 100 or as delivered by a user, a defined volume between a liquid sensor and a cut-off junction, and / or a defined volume between an overflow valve and a cut-off junction.

[0255] At block 608, a system composition may be stored. For example, the system composition may be stored in a blister device attached to the cartridge and / or a sealed chamber integrated into the cartridge body. The system composition may be used for lysis and / or hybridization. For example, the system composition may be stored in a blister device and transported to one or more extraction chambers 114 (e.g., the extraction chamber 114) for use in lysing the biological sample and / or hybridizing the nucleic acids of interest of the lysed biological sample.

[0256] In various examples, the system composition may comprise water, salt, a buffering compound, and / or surfactants. The system composition may include relatively high concentrations of salts. Types of salts found in the system composition may include, forAtty. Dkt. No.: 86357089example, KC1, MgCh and / or NaCl. In various examples, the surfactant may be Tween® 20. Further, the cartridge 100 may contain a certain volume of the system composition. For example, the cartridge 100 may include between 300 and 800 microliters of the system composition. For example, the cartridge 100 may include 500 microliters of the system composition. At block 610, the volume of the system composition may be metered so that the system composition volume used in the lysing and / or hybridization processes is at or around the predefined value. For example, 465 microliters of the specimen may be collected, and the volume may be metered to 450 microliters to be transported to and / or used in the extraction chamber 114.

[0257] At block 612, the biological sample may be lysed, stored with internal positive control (IPC), and mixed with system composition. For example, the metered biological sample may be transported to the extraction chamber 114. The system composition and the IPC may be added to the extraction chamber 114 prior to, concurrent with, and / or subsequent to addition of the biological sample to the extraction chamber 114. The IPC may be a lyophilized organism or synthetic organism. In various examples, the IPC may be a protein and / or organism present in a human sample. At block 628, the sample, the system composition, and the IPC may be mixed using sonication. For example, the lyse system 152 may be coupled with the extraction chamber 114. The sonicator 154 may perform sonication to mix the sample, buffer, and IPC.

[0258] In various examples, the extraction chamber 114 may be heated by an external heater and / or the sonicator 154. Sonication may heat the extraction chamber 114, mix the contents of the extraction chamber 114, and / or induce cavitation upon organisms (e.g., the IPC). In various examples, small beads or particles (e.g., ceramic beads, glass beads) may be included in the extraction chamber 114. The beads or particles may be agitated to mechanically lyse the solution. In various examples, lysis may result from one or more of heat, cavitation, and / or the lysing beads.

[0259] After lysis to process 612, a first set of paramagnetic particles 116 are added to the extraction chamber 114.

[0260] Referring now to block 614, a series of processes for preparing a sample for a PC A reaction are shown. The processes included in the block 614 may be repeated in series n number of times to prepare n number of samples for PCA reactions. In the method 600, three sampleAtty. Dkt. No.: 86357089preparations are shown and described. However, it should be understood that any number of sample preparations may be performed.

[0261] At process 616, a common lysate may be used for all n samples that are prepared. For example, at process 612, lysing the sample with IPC and the system buffer may produce a lysate. The same lysate produced at process 612 may be used in preparation of all n samples.

[0262] At process 618, potential targeted nucleic acids in the solution lysed at block 612 may be hybridized to the paramagnetic particles (e.g., magnetic particles 116). In the extraction chamber 114, the lysate may reconstitute lyophilized paramagnetic particles. The lyophilized paramagnetic particles may be functionalized with capture oligonucleotides that may be designed to capture RNA and / or DNA strands from target organisms. The oligonucleotides may be utilized as a half of a primer pair during amplification (e.g., during the PCA amplification reaction).

[0263] During hybridization, the magnetic particles 116 may be mixed and agitated with the lysate. Mixing and agitation may optimize a capture efficiency of the target nucleic acids. In various examples, hybridization may occur between 55 and 65 degrees Celsius. For example, hybridization may occur at 62 degrees Celsius. In various examples, hybridization may occur with a high salt content (e.g., MgCh or NaCl). Mixing may occur, for example, by reciprocating flow between two chambers (e.g., a first extraction chamber 114 and a second extraction chamber 114) such that vortices form at an entrance of each chamber to mix the lysate with the magnetic particles 116.

[0264] After hybridization, the hybridized magnetic particles 116 are held or trapped to a side of the extraction chamber 114 via, for example, an applied magnetic field generated by a magnetic field generator 156. While the hybridized magnetic particles 116 are trapped to the side or wall of the extraction chamber 114, the lysate (e.g., the IPC and the system composition) is transferred to a storage chamber (e.g., the lysate storage chamber 168). In various implementations, the lysate is stored and transported through the cartridge such that the lysate does not contact any surface that may also contact the master mix reagent.

[0265] At block 620, the wash buffer (e.g., wash buffer 110) may be stored. For example, the wash buffer 110 may be stored in a blister device attached to the cartridge and / or a sealed chamber integrated into the cartridge body. The wash buffer 110 may be used, at block 622, for washing magnetic particles (e.g., magnetic particles 116) and / or the extraction chamberAtty. Dkt. No.: 86357089114. For example, at block 620, the wash buffer 110 may be stored in a blister device and transported to an extraction chamber 114 for use in washing, at block 622, the magnetic particles 116 trapped to a wall of the extraction chamber 114 to prepare for amplification and detection of the nucleic acids of interest of the biological sample. The wash may resuspend the hybridized magnetic particles 116 in the second wash buffer within the extraction chamber 114. In some examples, resuspension is performed via sonication.

[0266] In various examples, the wash buffer 110 may comprise water, salt, a buffering compound, and / or surfactants. The wash buffer 110 may include moderately high concentrations of salts (e.g., relative to the salt concentrations in the system composition). Types of salts found in the wash buffer 110 may include, for example MgCh and / or NaCl. In various examples, the surfactant may be Tween® 20. The wash buffer 110 may also include KC1. Further, the cartridge 100 may contain a certain volume of the wash buffer 110. For example, the cartridge 100 may include between 100 and 400 microliters of the wash buffer 110. For example, the cartridge 100 may include 250 microliters of the wash buffer 110. In various examples, the cartridge 100 may include a plurality of wash buffers 110, each having a different composition and / or different uses. For example, a first wash buffer may include a stronger washing agent, and a second wash buffer may be milder. For example, the first wash buffer 110 may include 0.05 M tris buffer, 0.15 M NaCl, 0.0025 M KC1, and 0.05% Tween® 20, while the second wash buffer 110 may include 0.025 M tris buffer, 0.05 M NaCl, 0.0015 M KC1, and 0.01% Tween® 20. The first wash buffer may be used to wash the extraction chamber 114 and suspend hybridized magnetic particles 116, and the second wash buffer may be used to rinse the extraction chamber 114 and transfer the hybridized magnetic particles to the detection chamber 120.

[0267] In some implementations, a second wash of the extraction chamber 114 and the hybridized magnetic particles 116 is performed using a second wash buffer 110. The second wash may be performed using the same steps as are performed for the first wash. The second wash may trap the hybridized magnetic particles 116 and any exchanging fluids in the extraction chamber 114. The hybridized magnetic particles 116 that have been resuspended in the first or second wash buffer 110 are transferred to a first detection chamber 120 (e.g., a PCA chamber).

[0268] At block 624, the magnetic particles 116 are trapped onto a heating element (e.g., the heating element 122) in the first detection chamber 120 (e.g., a PCA chamber). The heatingAtty. Dkt. No.: 86357089element 122 may be or include, in various examples, a continuous, structured, or shaped metal foil, metal wires, a conductor and / or resistor layer deposited and / or plated, and / or backed by a heat spreader. In various examples, in order for the PCA process to occur, the paramagnetic particles with the captured nucleic acids may be concentrated at a thermocycling zone of the heating element. Thus, after hybridization, the hybridized magnetic particles suspended in wash buffer may be transported to the first detection chamber 120, where the particles are “trapped” or otherwise attached to the heating element 122.

[0269] The hybridized magnetic particles may be delivered to the detection chamber 120. An external permanent magnet (e.g., magnetic field generator 156) may create a magnetic field. The generated magnetic field may attract the paramagnetic particles onto a surface of the heating element 122. In various examples, the magnetic particles may be distributed uniformly across the heating element 122. Further, in various examples, a flow of the lysate may be continuous, in discrete steps, or any combination thereof.

[0270] At block 626, the PCA buffer may be stored. For example, the PCA buffer may be stored in a blister device attached to the cartridge and / or a sealed chamber integrated into the cartridge body. The PCA buffer may be used to reconstitute a master mix reagent used for reverse transcription and PCA amplification (e.g., the amplification process). For example, the PCA buffer may be stored in a blister device and transported to one or more detection chambers 120 (e.g., a PCA chamber) for use preparing for amplification of the nucleic acids of interest.

[0271] In various examples, the PCA buffer may comprise water and / or salt. The PCA buffer may include small concentrations of salts (e.g., relative to the salt concentrations in the system composition and / or the wash buffer 110). Types of salts found in the PCA buffer may include, for example MgCh. In various examples, the PCA buffer may include a surfactant (e.g., Tween® 20) and / or a buffering compound. Further, the cartridge 100 may contain a certain volume of the PCA buffer. For example, the cartridge 100 may include between 100 and 400 microliters of the PCA buffer. For example, the cartridge 100 may include 200 microliters of the PCA buffer.

[0272] At block 628, the volume of the PCA buffer may be metered so that the PCA buffer volume used in the reconstitution process is at or around the predefined value. For example, 235 microliters of the specimen may be collected, and the volume may be metered to 60 microliters to be transported to and / or used in the detection chamber 120. Metering the PCAAtty. Dkt. No.: 86357089buffer may control a concentration of the subsequently reconstituted master mix reagent, which may ensure proper amplification of the nucleic acids of interest. In various examples, the PCA buffer may be metered by pumping the PCA buffer through a defined volume between a liquid sensor and a fluidic T-junction bisecting the PCA buffer. Air may then be pumped into the T-junction such that a defined volume of the PCA buffer is further transported.

[0273] At block 630, a PCA master mix reagent may be reconstituted. A master mix may be used for reverse transcription, PCA amplification, and / or real-time fluorescence detection. The master mix may be lyophilized (e.g., freeze dried). The PCA master mix reagent may be stored, for example, in a lyophilized pellet or cake, an air-dried pellet or cake, and / or sealed with a plastic plug or film.

[0274] In various examples, the PCA master mix reagent may include a plurality of active ingredients, such as: reverse transcriptase, polymerase, and dNTPs. For each reaction target, the master mix reagent may include a set of primers (e.g., one half of a primer pair used in PCR) and a fluorescent probe. In various examples, lyophilization excipients may include sugars, such as, for example, trehalose.

[0275] In various examples, a metered amount of the master mix may be used to reconstitute the master mix reagent. The master mix reagent may be lyophilized. The PCA buffer may be mixed with the master mix by reciprocally pumping between a storage chamber storing the original master mix reagent and a channel leading to the chamber.

[0276] At block 632, the detection chamber 120 is loaded with the reconstituted master mix reagent. In order for amplification to be performed, the master mix reagent may be located in the detection chamber 120 where temperature incubation and / or thermocycling occurs. In various examples, air bubbles may interfere with optical detection of the nucleic acid of interest. Thus, the system may remove any air bubbles in the detection chamber 120 after the master mix reagent is loaded. Air bubbles may be managed, for example, by air bubble traps with stagnant chamber geometries, hydrophobic vent membranes over a channel or chamber, columns, posts, filters, elongated vertical chambers for buoyant bubble collection, etc. In various examples, air may be minimized to reduce movement when the solution is heated. Specifically, air on at least one send of the reaction solution may be minimized. For example, a valve may be positioned on one or more entrances and / or exits to the detection chamber 120 to remove air from the detection chamber 120.Atty. Dkt. No.: 86357089

[0277] Block 614 may conclude with process 632. At block 634, the processes performed in block 614 (e.g., blocks 616-632) are performed to prepare a first sample. That is, a first sample is prepared and delivered to a first detection (e.g., PCA) chamber 120. At block 636, the processes performed in block 614 (e.g., blocks 616-632) are performed to prepare a second sample. That is, a second sample is prepared and delivered to a second detection (e.g., PCA) chamber 120. At block 638, the processes performed in block 614 (e.g., blocks 616-632) are performed to prepare a third sample. That is, a third sample is prepared and delivered to a third detection (e.g., PCA) chamber 120. Blocks 634-638 may be performed sequentially or serially.

[0278] At block 640, amplification is performed. In various examples, amplification may be or include a PCA reaction, as will be described in greater detail with respect to FIG. 7. The PCA reaction may be a low-plex PCA reaction. In various examples, an amplification reaction may be performed in each of the first, second, and third PCA chambers in parallel. Upon amplification, a real-time multi-channel detection may be performed to detect the nucleic acids of interest. For example, the optical unit 162 may detect an absence, presence, and / or amount of the nucleic acid of interest. In various examples, detection may be performed using optical fluorescence and / or electrochemical detection with functionalized surfaces.

[0279] At block 642, the results of the amplification may be analyzed and presented. For example, the results may be analyzed to determine a presence, absence amount, etc. of the nucleic acid of interest. The results may be displayed, for example, via a user interface.

[0280] Referring now to FIG. 7, a method 700 for pulse controlled amplification (PCA) is shown, according to an example implementation. PCA may be performed to amplify the one or more nucleic acids of interest to be able to be detected. In various examples, PCA may be performed in one or more components of the cartridge 100, such as the extraction chamber 114 and / or the detection chamber 120. In various examples, a method other than PCA may be utilized to amplify the one or more nucleic acids of interest. For example, PCR may be used to amplify the nucleic acids.

[0281] At block 702, the lysate containing the nucleic acid of interest is hybridized to capture oligonucleotides. Hybridization may occur in the extraction chamber 114. After hybridization, the nucleotides of interest and the capture oligonucleotides may be transported to the detection chamber 120. The magnetic particles 116 with the capture oligonucleotides are shown in FIG.7 as magnetic particles with capture oligonucleotides 714. In the detection chamber, theAtty. Dkt. No.: 86357089magnetic particles may be docked to the heating element 122, shown in FIG. 7 at element 716. For example, the magnetic field generator 156 may generate a magnetic field such that the magnetic particles 116 dock to the heating element 122.

[0282] At block 704, the capture oligonucleotides attached to the docked magnetic particles may undergo elongation to increase a length of the strand or strands of the capture oligonucleotides. For example, if the capture oligonucleotide is RNA, the capture oligonucleotide may undergo reverse transcription. If the capture oligonucleotide is DNA, the capture oligonucleotide may undergo elongation. Transcription or reverse transcription and / or elongation may occur when the detection chamber 120 is at an annealing or elongation temperature (e.g., between 50 and 80 degrees Celsius). For example, the annealing or elongation temperature may be 72 degrees Celsius.

[0283] At block 706, an electrical pulse 718 may be delivered through the heating element 122. The electrical pulse may generate heat such that a heating zone having the local reaction liquid is heated to a predefined temperature value The predefined temperature value may be, for example, within a range of around 90 to 105 degrees Celsius. For example, the liquid may be heated to 100 degrees Celsius. The predefined temperature value may be a denaturation or melting temperature at which the DNA or RNA denatures. The local reaction liquid may be a portion of the total volume of liquid in the detection chamber 120. For example, the local reaction liquid may be between less than 1% and 5% of the total liquid volume in the reaction chamber. In various examples, the local reaction liquid may be liquid surrounding the magnetic particles 116, the capture oligonucleotides, the nucleic acids of interest, etc. that undergo a reaction / amplification. During heating of the heating element 122 and the local reaction liquid, the capture oligonucleotides attached to the magnetic particles 116 may denature. As a result, the target oligonucleotides may become free in solution. In various examples, the heating zone may quickly return to an annealing or elongation temperature value from the denaturation temperature value. For example, due to a heat capacity of the detection chamber 120, the locally heated area or volume of the detection chamber 120 may rapidly decrease. During pulse delivery (e.g., while the heating element 122 heats a portion of the reaction chamber), temperature changes may occur at a rate greater than 10.000 degrees Celsius per second. For example, the temperature may change at 11.000 degrees Celsius per second.

[0284] At block 708, primers 720 may bind to single strand oligonucleotides on the magnetic particles (e.g., the primers anneal to the denatured oligonucleotides). The primers 720 are short,Atty. Dkt. No.: 86357089single-stranded segments of nucleic acid (e.g., DNA) that are designed to be complementary to the beginning and / or end of the target sequence that will be amplified (e.g., the nucleic acid of interest). In some examples, the primers 720 may be forward and / or reverse primers (e.g., denoting a direction of elongation during the polymerization by the polymerase enzyme). In some examples, the primers may be forward and / or reverse primers. Forward and reverse primers may denote a direction of elongation during the polymerization by the polymerase enzyme. The primers 720 are used during the amplification and / or elongation steps of the reaction and may be part of the master mix composition. In some examples, the primers 720 are complementary to the target oligonucleotide.

[0285] During the amplification / elongation step of the PCR, the primers 720 may bind to the nucleic acid of interest (e.g., the DNA sequence of interest) on each end of the sequence of interest that is to be amplified (e.g., the target nucleic acid is “bookended” by the primers). As will be described herein, enzymes (e.g., DNA polymerase 722) may copy the part of the target oligonucleotide sequence that falls between the primers, selectively amplifying the sequence of interest.

[0286] In various examples, the capture oligonucleotide may also be used as a primer. For example, if the capture oligonucleotide is being used as a reverse primer (e.g., relative directionality during elongation by the polymerase), then forward primers may be free in solution and may bind to the single strand oligonucleotide that are captured (by the capture oligonucleotide that also shares the function of the reverse primer). In other words, the annealing of two primers (as being described here) may first occur. One of the two primers may not be free in solution because it is also the capture oligonucleotide. As such, a free amplicon (e.g., a copy of the target nucleic acids) anneals itself to the capture oligonucleotide that also doubles as the reverse primer. The forward primer may be free in solution to also bind to the amplicon. The binding of the amplicon to the capture oligonucleotide may be the same as or similar to hybridization described above. Further, the reaction may have “forward” and “reverse” primers switch places, where the capture oligonucleotide functions as the forward primer and the free primer in solution is the reverse primer.

[0287] At block 710, the strand of the primers and the oligonucleotides may be elongated. For example, polymerase 722 may elongate the strand. Elongation of the single strand into a double strand may occur between forward and reverse primers attached to the double strand in the annealing step. Thus, elongation may occur between the forward primer and the captureAtty. Dkt. No.: 86357089oligonucleotide (which also functions as the reverse primer). During elongation, fluorophores may be released into the solution for detection of the amplified nucleic acids of interest. For example, Taqman probes may be used to release fluorophores into the solution. The optical unit 162 may detect the fluorophores to determine a corresponding value, absence, presence, etc. of the amplified nucleic acid of interest.

[0288] The processes described with respect to blocks 706-710 may be repeated. For example, after the strand has been elongated at block 710, another electrical pulse may occur at block 708. Thus, more oligonucleotides are again denatured, and the process may repeat to generate a plurality of the nucleic acids of interest, for example, a certain number of times. The number of times may be a predetermined number or may depend upon a number of amplified strands.

[0289] In various examples, a fraction of the released oligonucleotides may be recaptured by capture oligonucleotides on a functionalized magnetic particle. These oligonucleotides may be used for the cyclic amplification reaction, thus causing exponential replication of the target nucleotides.

[0290] FIG. 8A depicts an example blister device 800, in accordance with present implementations. As illustrated by way of example in FIG. 8A, the example blister device 800 can include at least a first rupturing member 810 (also referred to herein as a “piercing member”) to open or close a valve seal 812a (also referred to herein as a “one-time open valve”) of the blister device 800 (e.g., a portion of the lidding foil 860 adjacent with the rupturing member), a metal coated polymer film 820, a reagent storage cavity 830, a second rupturing member 840 to open or close valve seal 812b of blister device 800 (e.g., a portion of the lidding foil 860 adjacent the rupturing member), a first fluid input-output area 850, the lidding foil 860 at least partially corresponding to the reagent storage cavity 830, and a second fluid inputoutput area 870. One or more zones as discussed herein can comprise or include one or more blister devices similar to the blister device 800. This technical solution is not limited to blister devices discussed by way of example, and is not limited to exclude blister devices at any zone. The blister device 800 may be used to store liquid reagents in the cartridge 100. For example, the system composition and / or the wash buffer 110 may be stored in a blister device 800. The blister device 800 may be attached, coupled, or otherwise affixed to the cartridge 100. The blister device 800 can provide a technical improvement to achieve bidirectional flow between and through zones of a cartridge as discussed herein.Atty. Dkt. No.: 86357089

[0291] As shown in FIG. 8 A, the blister device 800 comprises rupturing members 810 and 840 that can be actuated to open the valve seals 812. The rupturing members 810 and 840 may be integrated into the metal coated polymer film 820. In various examples, the rupturing members 810 and 840 may be used to rupture one-time open valves (e.g., valve seals 812). One-time open valves 812 may minimize water loss out of the storage cavity 830 through the closed or sealed rupturing members 810 and 840. The rupturing members may allow a common pump (e.g., the syringe pump 402 of FIGS. 4A and 4B) to deliver the contents of the blister device 800 to the microfluidic network of the cartridge 100. For example, the fluid entering and exiting the blister device 800 may enter and / or exit one or both of the one-time open valves 812. Further, the valves 812 may allow the reagents stored in the blister device 800 to return to the blister device 800 after use. For example, a blister device 800 may store a wash buffer. The wash buffer may exit the blister device 800 (e.g., through a valve 812) for use in the hybridization or detection chamber to wash the nucleic acids of interest. After washing is complete, the wash buffer may reenter the blister device 800 through the valves 812a and / or 812b. Storing used liquid reagents in the original blister device 800 may reduce the use of additional waste chambers in or on the cartridge 100, which may reduce a cost of manufacturing the cartridge 100 and / or may reduce a size of the cartridge 100. As such, the blister device 800 may additionally function as a waste chamber to store a used or exhausted liquid reagent.

[0292] As described above, the blister device 800 may include one or more one-time open valves 812. The opening of the valves 812 may allow a pump of the cartridge to deliver the reagents 125 stored in a chamber or cavity 830 of the blister device to one or more locations of the cartridge. The valves may also allow the reagents to return to the chamber after use of the reagents. As will be described with respect to FIGS. 9A and 9B, the rupturing members 810 and 840 may interface with an actuator of the instrument 150 to modulate a compression of the one or more rupturing members .

[0293] The blister device 800 may be sealed off from channels and chambers of the cartridge 100 by the rupturing members 810 and 840 and / or the unruptured valves 812. The rupturing members 810 and 840 may cover a first port (e.g., the valve 812a and / or the first fluid inputoutput area 850) when the rupturing member is in a non-ruptured state. The blister device 800 may be in fluid communication with the channels and the chambers of the cartridge through the first port and / or via the first fluid input-output area 850 when the rupturing member is in a ruptured state.Atty. Dkt. No.: 86357089

[0294] The blister device 800 may comprise a second port (e.g., the valve seal 812b and / or an area underneath the rupturing member 840 and / or the second input output area 870) and may be sealed-off from the channels and the chambers of the cartridge by the second rupturing member. The blister device 800 may be in fluid communication with the channels and the chambers of the cartridge through the second port and / or via the second fluid input-output area 870 when said the rupturing member is in a ruptured state.

[0295] The blister device 800 may enclose a storage chamber when the storage chamber is configured in hermetically sealed-off relation from the channels and / or when the cartridge of the present disclosure is in a non-activated state. The storage chamber is configured for open communication with at least one of the plurality of zones and / or detection chambers such that liquid may flow freely between channels. For example, the blister device 800 may, when ruptured, allow fluid to flow freely between the blister device 800 and the extraction chamber 114.

[0296] In some examples, a first channel can be sealed-off from the blister device 800 by the valve seal 812a covering a first port when the first valve seal 812a is in a non-ruptured state. The first channel can be in fluid communication with the blister device 800 through the first port when the first valve seal 812a is in a ruptured state.

[0297] In some examples, a second channel can be sealed-off from the blister device 800 by the valve seal 812b covering the second port when the second valve seal 812b is in a nonruptured state. The second channel can be in fluid communication with the chamber through the second port when the second valve seal 812b is in a ruptured state.

[0298] The first and second valve seals 812a and 812b may be ruptured by the first and second rupturing members 810 and 840, respectively, to bring the first and second channels into fluid communication with the storage chamber through the first and second ports of the blister device 800. Upon selectively piercing, by the first and second rupturing members 810 and 840, at least one of the first and second valve seals 812a and 812b of the blister device 800 and forming the opening therein, the desired reagent can be introduced through a corresponding one of the channels having an opened port.

[0299] FIGS. 8B, 8C, and 8D depict a detailed view of the rupturing members 810 and / or 840 of the blister device 800 and the valve seals 812 of FIG. 8A, according to some examples. A chamber between the rupturing members 810 and / or 840 and the valve seals 812a and 812b,Atty. Dkt. No.: 86357089respectively, may include first and second fluid input-output areas 850 and 870. In various examples, the rupturing members 810 and / or 840 may be actuated, compressed, or otherwise manipulated to open a valve seal 812 (also referred to as a “one-time open valve” or a “valve”) and permit fluid flow. However, over-travel of the rupturing members 810 and 840 may cause a pressure restriction when liquid attempts to move through the blister device 800 and / in and out of the blister device 800. FIG. 8B depicts the valve seal 812 of the blister device 800 prior to actuation or travel of the rupturing member 810. The rupturing member 810 and the first input-output area 850 are shown. However, the elements of FIGS. 8B, 8C, and 8D may be the same or similar for rupturing member 840 and area 870. As shown in FIG. 8B, the rupturing member 810 may be uncompressed, and the valve seal 812a may be unbroken. The fluid contained in the blister device may be stored in a chamber of the blister device and may enter and exit the blister device from the input output area 850 via a channel 814. FIG. 8C shows the blister device 800 when the rupturing member 810 is not over traveled (e.g., the rupturing member 810 is only partially compressed). This may permit fluid to flow upon actuation of the rupturing member 810. FIG. 8D depicts the valve seal 812a of the blister device 800 after actuation, specifically after over-travel of the rupturing member 810. As shown in FIG. 8D, flow may be restricted due to over-travel of the rupturing member 810. For example, in FIG.8D, the rupturing member 810 has over traveled to reduce a size of the cavity 850, thus restricting fluid flow. As such, fluid may have difficulty traveling through the blister device 800 and to and from other components of the cartridge 100 via the channel 814.

[0300] FIG. 9A depicts an example blister system 900, in accordance with present implementations. As illustrated by way of example in FIG. 9A, an example blister system 900 can include at least a blister device 902 and one or more actuators 910 having stand offs 920 to control actuation depth, a contact geometry 930 to open a frangible seal of the blister device 902, an actuator 960, and an actuation direction 970. The blister device 902 may include frangible seals 940 (also referred to as a valve, e.g., the valve 812) and a reagent storage cavity 950. In various examples, the blister device 902 may be located in or on the cartridge 100. The actuator 910 may be located in or on the instrument 150. The actuator 910 (and / or 960) may be positioned in the instrument 150 such that the actuator aligns with the seals 940 of the blister device 902 to allow the actuator to break one or more seals of the blister device 902. The blister device 800 can correspond at least partially in one or more of structure and operation to the blister device 902, and can include or couple to one or more of the actuator 910, the stand offs 920, or the actuator 960 by one or more of the contact geometry 930 and the frangible sealsAtty. Dkt. No.: 86357089940 to operate the actuators 910 and 960 in the actuation direction 970. For example, the actuation direction 970 can correspond to a first direction of movement of the actuators 910 or 960 toward the frangible seals 940 according to a piercing operation of one or more rupturing members (e.g., the rupturing members 810 and 840) to open one or more of the frangible seals 940. For example, the actuation direction 970 can correspond to a second direction of movement of the actuators 910 or 960 away from the frangible seals 940 subsequent to a piercing operation to open one or more of the frangible seals 940. Various examples can comprise a blister device in accordance with this disclosure, such as (but not limited to) the examples of the blister devices depicted in FIGS. 8A-9B. The blister system 900 may be part of other devices (e.g., other cartridges and systems) that interface with other instruments and systems.

[0301] As discussed with respect to FIGS. 8A-8D, over-travel of the piercing elements (e.g., rupturing members) of the blister device 902 may cause flow restriction. The actuator 910 may prevent over constriction. As shown in FIGS. 9 A and 9B, the actuator 910 may include stand offs 920 and a contact geometry. The stand offs 920 and the contact geometry 930 may prevent over travel. For example, the stand offs 920 may extend further or be longer than the contact geometry. Thus, the stand offs 920 may prevent the contact geometry 930 from contacting the seals 940 at too great a depth, thus causing flow restriction.

[0302] FIG. 10A depicts an example system, in accordance with present implementations. As illustrated by way of example in FIG. 10 A, an example system 1000 A can include at least a diagnostic cartridge 1010, a diagnostic instrument 1020, and a dropper 1030 A. The diagnostic cartridge may be the same as or similar to the cartridge 100, and the diagnostic instrument 1020 may be the same as or similar to the instrument 150. Specifically, as will be described in greater detail herein, the cartridge may include a sample input chamber comprising a liquid port. For example, a biological sample may be inserted into the cartridge via the sample input chamber. The cartridge may include a liquid port via which liquid samples may be received.

[0303] The diagnostic cartridge 1010 can receive and interact with a sample fluid by one or more components thereof. For example, the diagnostic cartridge 1010 can include one or more portions corresponding to zones, chambers, channels, reservoirs, containers, or any combination thereof. Specifically, the diagnostic cartridge 1010 may include a plurality of zones. The plurality of zones may include an extraction zone having one or more extractionAtty. Dkt. No.: 86357089chambers and a detection zone having one or more detection chambers. Each zone of the plurality of zones may be in fluid communication with each other.

[0304] The diagnostic cartridge 1010 can include one or more contents corresponding to chemicals, biochemicals, liquids, solutions, powders, materials, or any combination thereof, that can be present at or transferable between one or more of portions of the diagnostic cartridge 1010. The diagnostic cartridge 1010 can include one or more components that can interact with one or more corresponding components of the diagnostic instrument 1020. For example, the diagnostic cartridge 1010 can include a heating element located in each detection chamber of the one or more detection chambers that can be placed in contact with or proximate to a heating element of the diagnostic instrument 1020, to heat content of a portion of the diagnostic cartridge 1010. The portion of the cartridge 1010 may be heated via an electrical connection to activate the one or more heating elements of each of the one or more detection chambers of the diagnostic cartridge 1010. The diagnostic cartridge 1010 can have a structure corresponding to a rectangular shape of a size corresponding to a palm. The diagnostic cartridge 1010 can have a height greater than a width of the diagnostic cartridge 1010, and a depth greater than a height of the diagnostic cartridge 1010.

[0305] For example, the diagnostic cartridge 1010 can be at least partially insertable into the diagnostic instrument 1020. That is, the cartridge 1010 may interface with the instrument. The instrument 1020 can include a sensor configured to detect the state of the cartridge or its contents. For example, the instrument 1020 can detect whether the cartridge 1010 is fully inserted into the diagnostic instrument 1020 by an optical sensor or the like. For example, the instrument 1020 can detect whether one or more portions of the cartridge are filled or ready for performance of a diagnostic test, by one or more sensors configured to detect fill level of one or more portions.

[0306] The diagnostic cartridge 1010 can couple with the dropper 1030 A via a sample input chamber (e.g., the sample input chamber ) disposed at a surface of the diagnostic cartridge 1010. The sample input chamber may include a liquid port to receive a liquid, such as the biological sample. The sample input chamber can be disposed at a top surface of the diagnostic cartridge 1010, when the diagnostic cartridge 1010 is oriented to be inserted into the diagnostic instrument 1020. For example, the cartridge 1010 is configured to couple to the instrument 1020 to provide a user interface presenting at least one of an indication of an amount of the sample fluid in the cartridge 1010 or a status of the diagnostic test performed by the cartridgeAtty. Dkt. No.: 863570891010. For example, the cartridge 1010 has a physical interface configured to couple the cartridge 1010 to a holding device configured to hold the cartridge 1010 upright during injection of the sample fluid.

[0307] The diagnostic instrument 1020 can interact with the diagnostic cartridge 1010 and can generate one or more indications corresponding to one or more interactions with the diagnostic cartridge 1010. The diagnostic instrument 1020 can include one or more electrical and electronic components to initiate, control, and / or stop one or more interactions, and to generate one or more indications. For example, the diagnostic instrument 1020 can include one or more sensor devices, motors, actuators, processors, displays, or any combination thereof. The diagnostic instrument 1020 can include a receptacle having a width corresponding to a width of the diagnostic cartridge 1010, and a height corresponding to a height of the diagnostic cartridge 1010. The diagnostic instrument 1020 can include one or more logical or electronic devices including but not limited to integrated circuits, logic gates, flip flops, gate arrays, programmable gate arrays, and the like. One or more electrical, electronic, or like devices, or components associated with the diagnostic instrument 1020 can also be associated with, integrated with, integrable with, replaced by, supplemented by, complemented by, or the like, a system processor or any component thereof. For example, an interaction can correspond to one or more chemical, biochemical, electrical, or electrochemical reactions corresponding to identification of one or more chemicals or biochemicals. One or more interactions can result in one or more changes in electrical, optical, chemical, or other characteristics and / or properties inside the cartridge. Such characteristics and / or properties can be detected with one or more sensors to, for example, identify a component (e.g., a molecule or microorganism), to determine a state of the component, to detect the component’s presence, and / or to determine the component’s quantity.

[0308] The diagnostic instrument 1020 can include a system processor that can execute one or more instructions associated with the system 1000, according to any of the depictions 1000A-C of the system 1000. The system processor can include an electronic processor, an integrated circuit, or the like including one or more of digital logic, analog logic, digital sensors, analog sensors, communication buses, volatile memory, nonvolatile memory, and the like. The system processor can include, but is not limited to, at least one microcontroller unit (MCU), microprocessor unit (MPU), central processing unit (CPU), graphics processing unit (GPU), physics processing unit (PPU), embedded controller (EC), or the like. The system processorAtty. Dkt. No.: 86357089can include a memory operable to store or storing one or more instructions for operating components of the system processor and operating components operably coupled to the system processor. The one or more instructions can include at least one of firmware, software, hardware, operating systems, embedded operating systems, and the like. The system processor or the diagnostic instrument 1020 generally can include at least one communication bus controller to effect communication between the system processor and the other elements of the system 1000.

[0309] The dropper 1030 A can store contents and expel contents to the diagnostic cartridge 1010. For example, the dropper 1030A can have a cylindrical shape including an inlet at a first end and an outlet at a second end. The dropper 1030 A can include a reservoir corresponding to the cylindrical shape. For example, the dropper 1030A can have a reservoir with a volume corresponding to a medical syringe. The inlet of the dropper 1030 can correspond to a cap that can be opened to allow filling of the reservoir with contents. The outlet of the dropper 1030 A can correspond to a nozzle. For example, the nozzle of the dropper 1030A can have a shape that can be mated with the sample input chamber of the diagnostic cartridge 1010. The dropper 1030A can be in a state disconnected from or not mated with the diagnostic cartridge 1010. For example, the dropper 1030A can be in a state prior to or subsequent to filling of the diagnostic cartridge 1010 with the content of the dropper 1030 A.

[0310] FIG. 10B depicts an example system, in accordance with present implementations. As illustrated by way of example in FIG. 10B, an example system 1000B can include at least a dropper 1030B operated by a user 1040 according to a direction 1050. The dropper 1030B can correspond at least partially in one or more of structure and operation to the dropper 1030 A. For example, the dropper 1030B can be in a state corresponding to filling of the diagnostic cartridge 1010 with the content of the dropper 1030 A. The user 1040 can correspond to an individual operating one or more of the dropper 1030B, the diagnostic cartridge 1010, and the diagnostic instrument 1020. For example, the user 1040 can orient the dropper 1030B to align the sample input chamber of the diagnostic cartridge 1010 to face the outlet of the dropper 1030B. For example, the user 1040 can move one or more of the diagnostic cartridge 1010 and the dropper 1030B in the direction 1050 to mate the inlet of the diagnostic cartridge 1010 with the outlet of the dropper 1030B.

[0311] FIG. 10C depicts an example system, in accordance with present implementations. As illustrated by way of example in FIG. 10C, an example system 1000C can include at least aAtty. Dkt. No.: 86357089display device 0. The display device 0 can present one or more indications of one or more biochemical characteristics associated with a sample fluid, and can include an electronic display. An electronic display can include, for example, a liquid crystal display (LCD), a lightemitting diode (LED) display, an organic light-emitting diode (OLED) display, or the like. The display device 0 can receive, for example, capacitive or resistive touch input. The display device 0 can be housed at least partially within the diagnostic instrument 1020. The display device 0 can present one or more indications via one or more user interfaces that can include one or more graphical presentations and graphical control affordances. For example, a control affordance can include a portion of a user interface configured to detect user input. Example I / O components thus include, without limitation, a touchscreen display, a keypad or keyboard, biometric sensors such as a fingerprint scanners, buttons, switches, computer mice, microphones (e.g., for voice inputs such as test commands (e.g., “begin test”), passcodes (e.g., passcode known to authorized user), and / or voice recognition (e.g., analysis of voice signature of a user to record identity and / or compare voice signature for authentication or identity confirmation), speakers (e.g., for status updates such as “test in progress” or “test complete” or for speaking instructions for how to use an instrument or cartridge such as “insert cartridge further”), and / or other input / output devices.

[0312] In various examples, the diagnostic cartridge 1010 may include one or more extraction chambers (e.g., extraction chambers 114), one or more detection chambers (e.g., detection chamber 120 one or more reagents (e.g., reagents 125), and a plurality of magnetic particles (e.g., magnetic particles 116).

[0313] At least one of the one or more extraction chambers is a hybridization chamber to hybridize the one or more nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles. Further, at least one of the one or more detection chambers is an amplification chamber and one or more heating elements of the amplification chamber is a foil to interact with an electrical connection of an instrument to provide heat modulation to amplify the one or more nucleic acids of interest that have been hybridized to the at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles.

[0314] The diagnostic cartridge may include a transparent window in at least one detection chamber of the one or more detection chambers to allow an optical unit in communication withAtty. Dkt. No.: 86357089the at least one detection chamber to detect a plurality of amplification products indicative of a presence, absence, or amount of the plurality of amplified nucleic acids of interest.

[0315] The one or more extraction chambers may be used to lyse the biological sample to release one or more nucleic acids of interest from the biological sample. In various examples, one of the one or more extraction chambers may be a hybridization chamber to hybridize the one or more nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles.

[0316] Each of the one or more detection chambers may include a heating element at side portion of the detection chamber. The heating element may include a plurality of layers. For example, a layer of the heating element may be a foil. In various examples, the plurality of layers may further include a heat spreading element and / or an adhesive.

[0317] At least one of the one or more detection chambers may be an amplification chamber configured to amplify the nucleic acids of interest. One or more heating elements of the amplification chamber may be a foil to interact with an electrical connection of an instrument to provide heat modulation to amplify the one or more nucleic acids of interest that have been hybridized (e.g., in the extraction chambers 114) to the at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles.

[0318] The cartridge may further include one or more blister devices. Each blister device may store reagents (e.g., the wash composition). Each of the one or more blister devices may include one or more one-time open valves to allow a pump of the cartridge to deliver the reagents stored in a chamber of the blister device to one or more locations of the cartridge. The valves may also allow the reagents to return to the chamber after use of the reagents. The one or more onetime open valves may interface with an actuator of an instrument configured to modulate travel of a piercing element to open a valve or seal.

[0319] FIG. 11A depicts an example cartridge environment, in accordance with present implementations. As illustrated by way of example in FIG. 11 A, an example cartridge environment 1100 A can include at least a lower fill indication member 1110, an upper fill indication member 1112, a face 1120, a side portion 1130, and a view window 1140A. The cartridge environment 1100 A can correspond to a first state of a portion 1100 of the system 1000 according to one or more of systems 1000A-C as illustrated by way of example in FIG.10A-C.Atty. Dkt. No.: 86357089

[0320] The lower fill indication member 1110 can correspond to a physical component of the diagnostic cartridge 1010. The lower fill indication member 1110 can be located at a position that indicates a minimum fill level of a liquid with respect to the view window 1140A. For example, the lower fill indication member 1110 can indicate whether an amount of sample fluid from the dropper 1030B meets or exceeds a first vertical level in the view window 1140 A with respect to a particular diagnostic test. For example, the lower fill indication member 1110 can be formed or placed at a location over the view window 1140 A along a vertical direction of the view window 1140A to indicate a minimum amount of fluid corresponding to a particular diagnostic test. For example, the lower fill indication member 1110 can be formed as a component of the diagnostic cartridge 1010 at a first vertical position to indicate a minimum amount of fluid corresponding to a COVID-19 diagnostic test. For example, the lower fill indication member 1110 can be formed as a component of the diagnostic cartridge 1010 at a second vertical position to indicate a minimum amount of fluid corresponding to a seasonal flu diagnostic test. For example, the cartridge 1010 can include the predetermined position corresponding to a minimum amount of the sample fluid needed for performance of the diagnostic test.

[0321] The upper fill indication member 1112 can correspond to a physical component of the diagnostic cartridge 1010 distinct from the lower fill indication member 1110. The upper fill indication member 1112 can be located at a position that indicates a maximum fill level of a liquid with respect to the view window 1140 A. For example, the upper fill indication member 1112 can indicate whether an amount of sample fluid from the dropper 1030B meets or exceeds a second vertical level in the view window 1140 A with respect to a particular diagnostic test. For example, the upper fill indication member 1112 can be formed or placed at a location over the view window 1140A along a vertical direction of the view window 1140A to indicate a maximum amount of fluid corresponding to a particular diagnostic test. For example, the upper fill indication member 1112 can be formed as a component of the diagnostic cartridge 1010 at a third vertical position to indicate a maximum amount of fluid corresponding to a CO VID-19 diagnostic test. For example, the upper fill indication member 1112 can be formed as a component of the diagnostic cartridge 1010 at a fourth vertical position to indicate a maximum amount of fluid corresponding to a seasonal flu diagnostic test.

[0322] The face 1120 can at least partially frame view window 1140 A. The face 1120 can at least partially integrate with or connect with one or more of the lower fill indication memberAtty. Dkt. No.: 863570891110 and the upper fill indication member 1112. For example, the face 1120 can be formed to include a portion of a component of the diagnostic cartridge 1010 defining an opening at least partially surrounding the view window 1140 A. For example, the face 1120 can be formed with one or more of the lower fill indication member 1110 and the upper fill indication member 1112. For example, the face 1120, the lower fill indication member 1110, and the upper fill indication member 1112 can be integrally formed of a single solid piece. For example, the single solid piece can include or be a stiff polymer or plastic. The face 1120 can be oriented according to a face plane that intersects a top plane corresponding to the top surface of the diagnostic cartridge 1010 and a front plane corresponding to the front surface of the diagnostic cartridge 1010. For example, the face plane can be oriented at a 45 degree angle with respect to one or more of the top plane and the front plane. Thus, the face 1120 can provide a technical improvement to increase visibility of a view window 1140A during a filling process of the diagnostic cartridge 1010.

[0323] The side portion 1130 can correspond to a component of the diagnostic cartridge 1010 covering at least a portion of the diagnostic cartridge 1010. For example, the side portion 1130 can be integrally formed with one or more of the lower fill indication member 1110, the upper fill indication member 1112, and the face 1120. For example, the face 1120, the lower fill indication member 1110, and the upper fill indication member 1112 can be integrally formed of a single solid piece with the side portion 1130.

[0324] The view window 1140A can correspond to at least a portion of a reservoir of the diagnostic cartridge 1010 that aligns with an opening in the face 1120. For example, the reservoir of the diagnostic cartridge 1010 can be disposed along the face plane to render visible at least a portion of side wall through which a fill level of a liquid in the reservoir. For example, at least the portion of the reservoir aligned with the opening of the face 1120 can include or be a transparent or translucent material. The view window 1140 A can correspond to a state of a view window 1140 having no fluid visible therethrough. For example, the view window 1140A can frame a reservoir of the diagnostic cartridge 1010 absent any fluid or containing an amount of fluid below a threshold of visibility in the view window 1140A.

[0325] FIG. 11B depicts an example cartridge environment, in accordance with present implementations. As illustrated by way of example in FIG. 11B, an example cartridge environment 1100B can include at least a view window 1140B, and a fluid 1150 visible from view window 1140B.Atty. Dkt. No.: 86357089

[0326] The view window 1140B can correspond at least partially in one or more of structure and operation to the view window 1140B. The view window 1140B can correspond to a state of a view window 1140 having fluid 1150 visible therethrough. For example, the view window 1140B can frame a reservoir of the diagnostic cartridge 1010 containing an amount of liquid visible in the view window 1140A. For example, the fluid 1150 visible from view window 1140B can be at a level above the lower fill indication member 1110 and below the upper fill indication member 1112. Thus, the lower fill indication member 1110 and the upper fill indication member 1112 can provide a technical improvement to increase visibility of fluid 1150 deposited to the diagnostic cartridge 1010 during depositing of the fluid 1150 by the user 1040.

[0327] FIG. 12A depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 12 A, an example cartridge environment 1200 A in a cross-sectional view can include at least an upper portion 1210, a lower portion 1212, a reservoir 1220, a fluid 1230 A in the dropper 1030B, a fluid 1232A in the reservoir 1220, and an air gap 1234A between the dropper 1030B and the reservoir 1220. The cartridge environment 1200A can correspond to a first filling state including the diagnostic cartridge 1010 and the dropper 1030B. For example, the first filling state can correspond to a start of a transfer of fluid 1230 A from the dropper 1030B to the diagnostic cartridge 1010.

[0328] The upper portion 1210 can correspond to a portion of the reservoir 1220 structured to couple with or mate with the outlet of the dropper 1030B. For example, the upper portion 1210 can define an opening extending from a top surface of the diagnostic cartridge 1010 to the reservoir 1220. For example, the upper portion 1210 can define an opening having a cross section corresponding to a cross section of the outlet of the dropper 1030B. For example, the opening of the upper portion 1210 can have a circular cross section corresponding to a circular cross section of the dropper 1030B.

[0329] The lower portion 1212 can correspond to a portion of the reservoir 1220 structured to transport and store fluid. For example, the lower portion 1212 can extend in a direction corresponding to the face plane. For example, the lower portion 1212 can be integrally formed with the upper portion 1210 at an angle corresponding to an angle between the face plane and either the top plane or the front plane, or both. The position of the lower portion 1212 at the angle can result in the lower portion having a sloped surface 1214. The sloped surface 1214 ofAtty. Dkt. No.: 86357089the lower portion 1212 can be oriented to allow transport of the fluid 1232A without or mitigating dispersion of the fluid 1232A in the reservoir 1220. For example, the sloped surface 1214 can be oriented to prevent “breakage” of the fluid and to transport fluid in one body or a minimum number of bodies according to a surface tension of the fluid. For example, the sloped surface 1214 can be formed or oriented to achieve a technical improvement to prevent or minimize formation of separate droplets of the fluid 1232A. For example, the sloped surface 1214 can be formed or oriented to achieve a technical improvement to prevent or minimize formation of bubbles in the fluid 1232A. For example, the sloped surface 1214 can have an angle of 45 degrees with respect to the top plane of the diagnostic cartridge 1010, to mitigate dispersion of a fluid 1232A having a surface tension property corresponding, for example, to at least one of a liquid sample or a liquid sample mixed or otherwise combined with a transport medium. For example, a transport medium can correspond to or include a commercial transport medium, such as Copan UTM ®.

[0330] The reservoir 1220 can include both the upper portion 1210 and the lower portion 1212. For example, the reservoir 1220 can have a cylindrical shape enclosing a cavity that abuts the face 1120 and the view window 1140 A. For example, the reservoir 1220 can be attached with a body of the diagnostic cartridge 1010 and can include or be a transparent or translucent material. The reservoir 1220 can have one or more dimensions to achieve a technical improvement to eliminate or mitigate dispersion of the fluid 1232A in the reservoir 1220. For example, the reservoir 1220 can be formed with one or more of a particular inner depth 1222 and a particular outer depth 1224 corresponding to a surface tension property corresponding to at least one of a liquid sample or a liquid sample mixed or otherwise combined with a transport medium. For example, the inner depth 1222 can be between about 10 millimeters (mm) and about 20 mm, and can be about 15 mm. For example, the outer depth 1224 can be about 4 mm greater than the inner depth 1222. For example, the inner depth can correspond to a horizontal direction from a front of the reservoir 1220 at the view window 1140 A to a rear of the reservoir 1220 at a surface opposite to the front of the reservoir in the horizontal direction. For example, the horizontal direction can be parallel to the top plane of the diagnostic cartridge 1010. For example, the reservoir can have a depth ranging from about 5 mm to about 10 mm, and a width ranging from about 5 mm to about 10 mm. For example, the reservoir can correspond to a chamber. For example, the chamber can include a first surface coupled to the opening, and a second surface that is sloped with respect to the first surface such that the sample fluid flows down the second surface when the cartridge is upright. For example, the system can include aAtty. Dkt. No.: 86357089reservoir that has a depth between about 5 mm and about 10 mm and a width between about 5 mm and about 10 mm.

[0331] The fluid 1230 A in the dropper 103 OB can be transported between the dropper 1030B mated with the reservoir 1220. For example, the fluid 1230A can be expelled from the outlet of the dropper 1030B into the upper portion 1210 of the reservoir 1220 and onto the sloped surface 1214 of the lower portion 1212 of the reservoir 1220. The outlet of the dropper 1030B can be positioned at a distance from the sloped surface 1214 corresponding to a distance to mitigate or eliminate dispersion of the fluid 1232A in the reservoir 1220. The fluid can flow into the reservoir and fill the reservoir 1220 according to a fill line, with minimal or no dispersion that may render the fill level of the fluid 1232A difficult or impossible to detect visually. The air gap 1234A between the dropper 1030B and the reservoir 1220 can provide an outlet for air to escape the reservoir 1220 as it is replaced by the fluid 1232A. Thus, the air gap 1234A can provide the technical improvement of mitigating or preventing dispersion of the fluid 1232A by providing a pathway for air to flow that prevents or mitigates pressurization of or foaming of the fluid 1232A.

[0332] FIG. 12B depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 12B, an example cartridge environment 1200B in a cross-sectional view can include at least a fluid 1230A in the dropper 1030B, a fluid 1232B in the reservoir 1220, and an air gap 1234B between the dropper 1030B and the reservoir 1220. The cartridge environment 1200B can correspond to a second filling state including the diagnostic cartridge 1010 and the dropper 1030B. For example, the first filling state can correspond to a continuation of a transfer of fluid from the dropper 1030B to the diagnostic cartridge 1010, subsequent to the first filling state.

[0333] For example, the dropper 1030B can be removed from the inlet of the upper portion 1210 of the reservoir 1220 at any point during a filling process of the reservoir. However, the dropper 1030B does not need to be removed from the inlet of the upper portion 1210 to complete a filling process. The fluid 1230B can correspond to an amount of fluid less than an amount of the fluid 1230A. The fluid 1232B can correspond to an amount of fluid greater than an amount of the fluid 1232A. For example, the fluid 1232B can continue to flow into the reservoir 1220 with an absence of formation of bubbles or droplets. The air gap 1234B can be increased to the size of the inlet of the upper portion 1210 of the reservoir 1220 upon removal of the dropper 1030B from the inlet of the upper portion 1210.Atty. Dkt. No.: 86357089

[0334] FIG. 12C depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 12C, an example cartridge environment 1200C in a cross-sectional view can include at least a fluid 1230C in the dropper 1030B, and a fluid 1232C in the reservoir 1220. The cartridge environment 1200C can correspond to a third filling state including the diagnostic cartridge 1010 and the dropper 103 OB. For example, the third filling state can correspond to a continuation of a transfer of fluid from the dropper 1030B to the diagnostic cartridge 1010, subsequent to the second filling state. The fluid 1230C can correspond to an amount of fluid less than an amount of the fluid 1230A. The fluid 1232C can correspond to an amount of fluid greater than an amount of the fluid 1232B. For example, the fluid 1232C can continue to flow into the reservoir 1220 with an absence of formation of bubbles or droplets.

[0335] FIG. 12D depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 12D, an example cartridge environment 1200D in a cross-sectional view can include at least a fluid 1230D in the dropper 1030B, and a fluid 1232D in the reservoir 1220. The cartridge environment 1200D can correspond to a fourth filling state including the diagnostic cartridge 1010 and the dropper 1030B. For example, the fourth filling state can correspond to a continuation of a transfer of fluid from the dropper 1030B to the diagnostic cartridge 1010, subsequent to the third filling state. The fluid 1230D can correspond to an amount of fluid less than an amount of the fluid 1230C, or an absence of fluid in the dropper 1030B. The fluid 1232D can correspond to an amount of fluid greater than an amount of the fluid 1232C. For example, the fluid 1232D can complete a flow into the reservoir 1220 with an absence of formation of bubbles or droplets. For example, a level of the fluid 1232 can be visible through the view window 1140B according to a level corresponding to the fill level of the reservoir 1220.

[0336] FIG. 13 A depicts an example cartridge environment in plan view, in accordance with present implementations. As illustrated by way of example in FIG. 13 A, an example cartridge environment 1300 A in plan view can include at least a cartridge inlet 1310A, and an inlet cover 1320A. The cartridge inlet 1310A can correspond at least partially in one or more of structure and operation to the inlet of the upper portion 1210 of the reservoir 1220. The cartridge inlet 1310A can correspond to an open state that allows the outlet of the dropper 1030 A to mate with the cartridge inlet 1310A. In some examples, the inlet cover 1320A can slide from an openAtty. Dkt. No.: 86357089position to a closed position (e.g., similar to or the same as the sample input cover 303b). In some examples, the inlet cover can be a cap that can close or open over the cartridge inlet 1310A (e.g., similar to or the same as the sample input cap 303a). For example, the inlet cover 1320A can be at a position corresponding to the open state that allows the outlet of the dropper 1030A to mate with the cartridge inlet 1310A. For example, the cartridge can include a lid moveable to cover and seal the opening, the lid configured to permit the diagnostic test to proceed when covering the opening.

[0337] FIG. 13B depicts an example cartridge environment in plan view, in accordance with present implementations. As illustrated by way of example in FIG. 13B, an example cartridge environment 13006 in plan view can include at least a cartridge inlet 1310B, and an inlet cover 1320B. The cartridge inlet 1310B can correspond at least partially in one or more of structure and operation to the cartridge inlet 1310A. The cartridge inlet 1310B can correspond to a closed state that prevents or blocks the outlet of the dropper 1030 A from mating with the cartridge inlet 1310A, and prevents or blocks egress of fluid in the reservoir 1220. The inlet cover 1320B can correspond to a closed position, subsequent to a filling operation of the reservoir 1220. For example, the inlet cover 1320B can be at a position corresponding to the closed state that prevents or blocks the outlet of the dropper 1030 A from mating with the cartridge inlet 1310B.

[0338] This technical solution can provide at least a technical improvement to mitigate or eliminate contamination into an environment external to the cartridge 1010, by mitigating or eliminating dispersion of a sample fluid into an environment exterior to the cartridge 1010 via the cartridge inlet 1310. For example, the cartridge 1010 can transition between cartridge environments 1300A-B by movement of a cartridge at a predetermined velocity or within a range of predetermined velocities. For example, the inlet cover 1320A can move via a spring having a tension sufficiently low as to prevent a “snap” of the inlet cover 1320A into the depicted position of FIG. 13B. The “snap” can correspond to a speed of traversal of the inlet cover 1320 sufficient to cause ejection of at least a portion sample flid from the reservoir 1220 via the cartridge inlet 1310. For example, a speed of traversal can be greater than or equal to 0.5 seconds, but is not limited thereto. For example, the speed of traversal can be based on a spring coupling the inlet cover 1320A to the upper portion 1210 of the reservoir 1220.

[0339] For example, the technical solution can include the technical improvement to mitigate contamination into an environment external to the cartridge 1010, by a combination of structures of the cartridge 1010, to achieve the technical improvement of substantial mitigation,Atty. Dkt. No.: 86357089up to complete elimination, of dispersion of a sample fluid into an environment exterior to the cartridge 1010 via the cartridge inlet 1310. For example, the cartridge inlet 1310, corresponding to the upper portion 1210 of the reservoir 1220, can be structured to minimize dispersion by a structure to allow a tip of the dropper 1030 to be fully inserted into the upper portion 1210, with an allowance restricted to the air gap 1234A. Further, the inlet cover 1320 can be structured to mitigate or eliminate contact with sample fluid that has potentially been dispersed onto the top surface of the cartridge 1010. For example, the inlet cover 1320 can be structured to have a shape that covers the cartridge inlet 1310 including the cartridge inlet 1310 and a portion of the top surface of the cartridge 1010 within a predetermined distance of the cartridge inlet 1310. For example, the predetermined distance can correspond to a square or rectangular region of the top surface located surrounding the cartridge inlet 1310, as illustrated by way of example in Fig. 13 A.

[0340] FIG. 14 depicts an example cartridge panel, in accordance with present implementations. As illustrated by way of example in FIG. 14, an example cartridge panel 1400 can include at least a front portion 1420, and a top portion 1430. The panel 1400 can correspond to a single piece integrally formed to include the lower fill indication member 1110, the upper fill indication member 1112, the face 1120, the side portion 1130, to define the view window 1140. The face 1120 can include the lower fill indication member 1110, the upper fill indication member 1112, the face 1120, and the view window 1140. The top portion 1430 can include the cartridge inlet 1310, and the inlet cover 1320. For example, the cartridge inlet 1310 can be integrally formed with the top portion 1430, and the inlet cover 1320 can be attachable to the top portion 1430 to moveably slide at least from the open position to the closed position along the top portion 1430. The panel 1400 can achieve a technical improvement to provide a customizable cartridge indication via members 1110 and 1112 that can be formed to correspond to a particular diagnostic test, and can be attached to a diagnostic cartridge 1010 to customize the diagnostic cartridge 1010 to the diagnostic test and to customize the members 1110 and 1112 for the diagnostic test. This technical solution is not limited to the cartridge panel 1400. For example, at least one of the cartridge inlet 1310 or the inlet cover 1320 can be integrally formed with, integrated with, attached with, or coupled with the reservoir 1220.

[0341] FIG. 15 depicts an example user interface for cartridge environment, in accordance with present implementations. As illustrated by way of example in FIG. 15, an example user interface 1500 for a cartridge environment can include at least an environment presentationAtty. Dkt. No.: 863570891510, a diagnostic presentation 1520, and a reservoir presentation 1530. The user interface 1500 can be presented on or by the display device 0. For example, the system can include a device configured to securely couple with the cartridge and provide a user interface. The user interface 1500 can be configured to present an indication of a status of the diagnostic test performed by the cartridge.

[0342] The environment presentation 1510 can present a visual representation of one or more of the diagnostic cartridge 1010 and the diagnostic instrument 1020. For example, the environment presentation 1510 can present, at a first portion of the user interface, an indication of an arrangement of the diagnostic cartridge 1010 with respect to the diagnostic device 0. For example, the arrangement can correspond to an attachment or mating of the diagnostic cartridge 1010 with the diagnostic instrument 1020 by at least partially inserting the diagnostic cartridge in the diagnostic instrument 1020.

[0343] The diagnostic presentation 1520 can present a visual indication of a diagnostic test corresponding to the diagnostic cartridge 1010 or a diagnostic test corresponding to the diagnostic cartridge 1010 or the panel 1400. For example, the diagnostic presentation 1520 can present, at a second portion of the user interface, an indication of identifiers of the diagnostic test. For example, the identifiers can include an identification of one or more aspects of the test, the patient, the cartridge, or any combination thereof.

[0344] The reservoir presentation 1530 can present a visual indication of a fill level corresponding to a diagnostic test or the panel 1400 with respect to one or more of the member 1110 and 1112. For example, the reservoir presentation 1530 can include one or more members 1110 and 1112 present according to the member 1110 and 1112 on the panel 1400. For example, the reservoir presentation 1530 can present a visual indication including both the members 1110 and 1112, according to a panel 1400 that includes both the members 1110 and 1112. For example, the reservoir presentation 1530 can present a visual indication including only the member 1110, according to a panel 1400 that includes only the member 1110. For example, the reservoir presentation 1530 can present a visual indication including only the member 1112, according to a panel 1400 that includes only the member 1112. For example, the reservoir presentation 1530 can prompt the user to perform a filling operation according to a diagnostic test to level indicated by one or more of the members formed according to the diagnostic test. For example, the device can include where the one or more prompts correspondAtty. Dkt. No.: 86357089to injection of the sample fluid into the cartridge. For example, the device can include where the one or more prompts correspond to performance of the diagnostic test.

[0345] Aspects of this technical solutions disclosed herein may include a cartridge such as, in various examples, a cartridge consistent with the above disclosure. The cartridge may receive sample fluids to be tested. The cartridge may include components with structures to mitigate erroneous indications of amounts of sample fluid, during deposition of the sample fluid. A diagnostic testing architecture can include a single-use or limited-use cartridge that includes one or more contents that can interact with a sample fluid to perform a particular medical diagnostic by triggering one or more physical reactions with at least a portion of a sample fluid deposited at the cartridge. For example, contents can correspond to chemicals, liquids, or solids having particular chemical, biological, electrical, or mechanical properties, or any combination thereof. The diagnostic testing architecture can include a diagnostic testing device (e.g., an instrument) to detect the results or effects of one or more interactions with the sample fluid at the cartridge, and can determine and output one or more results corresponding to the results or effects of the one or more interactions. Thus, this technical solution can include one or more components including but not limited to one or more of a cartridge configured according to a particular diagnostic test, a dropper operable to deposit a sample fluid to the cartridge or a component thereof, and a diagnostic instrument to exchange signals with the cartridge and generate or output an indication corresponding to receipt of the sample fluid and / or to an interaction between the sample fluid and one or more portions of the cartridge.

[0346] At least one aspect is directed to one or more components that can be combined or assembled, for example, to form a cartridge configured to perform a particular diagnostic. For example, a cartridge can include one or more zones that can include one or more contents as discussed herein, according to a diagnostic to be performed. For example, a diagnostic can correspond to a medical diagnostic. For example, a medical diagnostic can include one or more tests for the presence of indicators of a molecule, microorganism, disease, or condition. In an example, a cartridge may comprise one or more diagnostic tests to detect indicators of one or more viruses (or components thereof) that can cause one or more diseases such as COVID-19, influenza, etc. The cartridge can include a body including one or more zones having one or more contents, and a panel portion including one or more indicators corresponding to a particular diagnostic. For example, a body having a reservoir integrated therewith can couple with a panel having a view window that can be aligned with a portion of the reservoir, inAtty. Dkt. No.: 86357089accordance with an attachment of the body with the panel. The panel can include one or more members that can be positioned at one or more locations relative to the reservoir to indicate various fill levels of the reservoir. The fill levels may indicate amounts of sample fluid suitable for performing a particular diagnostic. For example, the panel can be configured to include one or more of a minimum fill indicator and / or a maximum fill indicator that can align with the reservoir of the body and that can be positioned based on how much of a sample fluid is to be injected into the cartridge for the diagnostic. Thus, the panel can provide a technical solution to customize one or more indicators corresponding to a particular diagnostic.

[0347] At least one aspect is directed to a cartridge that can include a reservoir and an inlet structured to receive sample fluid, and to minimize or eliminate dispersion of the fluid in a manner that can result in an incorrect indication of sample fluid in the view window corresponding to the reservoir. For example, a user can input a sample fluid into a cartridge via an inlet located at an upper portion of the cartridge while concurrently viewing the view window and a portion of the reservoir aligned with the view window. The reservoir can include a transparent or translucent material to permit viewing of the sample fluid within the reservoir from the view window. The user can also view, at the view window, one or more members indicating at least one of a minimum fill level or a maximum fill level of the reservoir. The user can deposit sample fluid into the reservoir via the inlet while viewing the view window and the members to ensure that a fill level of the sample fluid in the reservoir appears above or below the members. This technical solution can accurately indicate a fill level of a reservoir during the deposit of the sample fluid, at least by a structure of the reservoir to minimize or eliminate dispersion of the sample fluid that may cause a fill level to appear dispersed or unclear. For example, formation of bubbles during depositing of a sample fluid can result in a layer of foam that may cause the volume of a fluid in the reservoir to appear incorrectly greater than its actual volume. Thus, this technical solution can provide at least a technical improvement to eliminate dispersion of a fluid during deposition to decrease risk of incorrect fluid sample deposit by a user to a cartridge, and thus reduce or eliminate waste of cartridge devices, biological specimens, or any combination thereof.

[0348] The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” “characterized by,” “characterized in that,” and variations thereof herein, is meant to encompass the items listed thereafter, equivalents thereof, and additionalAtty. Dkt. No.: 86357089items, as well as alternate implementations consisting of the items listed thereafter exclusively. In one implementation, the systems and methods described herein consist of one, each combination of more than one, or all of the described elements, acts, or components.

[0349] References to “or” may be construed as inclusive so that any terms described using “or” may indicate any of a single, more than one, and all of the described terms. References to at least one of a conjunctive list of terms may be construed as an inclusive OR to indicate any of a single, more than one, and all of the described terms. For example, a reference to “at least one of ‘A’ and ‘B’” can include only ‘A,’ only ‘B,’ as well as both ‘A’ and ‘B.’ Such references used in conjunction with "comprising" or other open terminology can include additional items. References to “is” or “are” may be construed as nonlimiting to the implementation or action referenced in connection with that term. The terms “is” or “are” or any tense or derivative thereof, are interchangeable and synonymous with "can be" as used herein, unless stated otherwise herein.

[0350] Directional indicators depicted herein are example directions to facilitate understanding of the examples discussed herein and are not limited to the directional indicators depicted herein. Any directional indicator depicted herein can be modified to the reverse direction or can be modified to include both the depicted direction and a direction reverse to the depicted direction, unless stated otherwise herein. While operations are depicted in the drawings in a particular order, such operations are not required to be performed in the particular order shown or in sequential order, and all illustrated operations are not required to be performed. Actions described herein can be performed in a different order. Where technical features in the drawings, detailed description or any claim are followed by reference signs, the reference signs have been included to increase the intelligibility of the drawings, detailed description, and claims. Accordingly, neither the reference signs nor their absence have any limiting effect on the scope of any clam elements.

[0351] The various ranges provided herein include the stated range and any value or sub-range within the stated range. Furthermore, when “about” is utilized to describe a value or percentage this includes, refers to, and / or encompasses variations (up to + / - ten %) from the stated value or percentage. In describing and claiming the examples disclosed herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.Atty. Dkt. No.: 86357089

[0352] Although specific examples have been illustrated and described herein, a variety of alternate and / or equivalent implementations may be substituted for the specific examples shown and described without departing from the scope of the present disclosure. This application is intended to cover any adaptations or variations of the specific examples discussed herein. For example, the method 200 may include additional operations not explicitly recited or may exclude certain recited operations in some examples. Various examples of the cartridge 100 or the instrument 150 may include additional components not explicitly recited, may exclude certain recited components, or may include the recited components in different relative positions than shown in the examples described above. Therefore, it is intended that the scope of this disclosure be limited only by the claims and the equivalents thereof.

Claims

Atty. Dkt. No.: 86357089WHAT IS CLAIMED IS:

1. A method for detecting a presence, absence, or amount of a nucleic acid of interest, the method comprising:inserting, into a cartridge, a fluid comprising a biological sample, wherein the cartridge comprises a plurality of zones comprising an extraction zone comprising one or more extraction chambers, each comprising a plurality of magnetic particles, and a detection zone comprising one or more detection chambers, each detection chamber comprising one or more heating elements, wherein each zone of the plurality of zones is in fluid communication with each other, and wherein the cartridge further comprises one or more reagents;lysing the biological sample into at least one of the one or more extraction chambers to release the one or more nucleic acids of interest from the biological sample, wherein lysing the biological sample produces a lysate;collecting a first set of hybridized magnetic particles, the first set of magnetic particles hybridized to a first type of nucleic acids of interest, by:hybridizing, into the at least one of the one or more extraction chambers, the first type of nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles, the at least one capture oligonucleotide specific to the first type of nucleic acids of interest;holding the first set of hybridized magnetic particles on a side of the extraction chamber;transporting the lysate to a waste storage chamber;suspending the first set of hybridized magnetic particles in the at least one of the one or more extraction chambers with a first wash buffer; andtransporting the first set of hybridized magnetic particles to a first detection chamber of the one or more detection chambers;collecting, using the lysate, a second set of hybridized magnetic particles, the second set of magnetic particles hybridized to a second type of nucleic acids of interest;amplifying each of the first and second sets of hybridized magnetic particles in at least one of the plurality of zones, via an amplification reaction, to provide a plurality of the first type of the nucleic acid of interest and the second type of the nucleic acid of interest; and detecting a plurality of amplification products indicative of the presence, absence, or amount of the plurality of the first and second types of nucleic acids of interest via an opticalAtty. Dkt. No.: 86357089unit in communication with each of the first and second detection chambers of the one or more detection chambers.

2. The method of claim 1, further comprising:after collecting the second set of hybridized magnetic particles:transferring a reconstituted first master mix reagent to the at least one detection chamber of the one or more detection chambers;delivering a plurality of amplification reagents unique to the first set of hybridized magnetic particles to the at least one detection chamber;transferring a reconstituted second master mix reagent to a second detection chamber of the one or more detection chambers, the second set of hybridized magnetic particles to be delivered to the second detection chamber of the one or more detection chambers; anddelivering a plurality of amplification reagents unique to the second set of hybridized magnetic particles to the second detection chamber.

3. The method of claim 1, wherein collecting the first set of hybridized magnetic particles further comprises:transporting the lysate from the waste storage chamber back to the at least one of the one or more extraction chambers after transporting the first set of hybridized magnetic particles to the first detection chamber of the one or more detection chambers, and before collecting the second set of hybridized magnetic particles.

4. The method of claim 1, wherein the magnetic particles are held to the side of the extraction chamber in an applied magnetic field.

5. The method of claim 1, further comprising:returning the first wash buffer to a storage blister device subsequent to suspending the first set of hybridized magnetic particles; andwashing the first set of hybridized magnetic particles with a second wash buffer.

6. The method of claim 5, further comprising suspending the first set of hybridized magnetic particles in the at least one of the one or more extraction chambers via the secondAtty. Dkt. No.: 86357089wash buffer, wherein the first set of hybridized magnetic particles is transported to the first detection chamber via the second wash buffer.

7. The method of claim 1, wherein collecting the first set of hybridized magnetic particles further comprises trapping the first set of hybridized magnetic particles within a threshold distance of the one or more heating elements of the first detection chamber.

8. The method of claim 1, wherein collecting the second set of hybridized magnetic particles comprises:hybridizing, into the at least one of the one or more extraction chambers, the second nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles, the at least one capture oligonucleotide specific to the second type of nucleic acid of interest;holding the second set of hybridized magnetic particles on a side of the extraction chamber;transporting the lysate to a lysate storage chamber;suspending the second set of hybridized magnetic particles in the at least one of the one or more extraction chambers with a third wash buffer, the third wash buffer of a same type as the first wash buffer;transporting the second set of hybridized magnetic particles to a second detection chamber of the one or more detection chambers;trapping the hybridized plurality of magnetic particles within a threshold distance of the one or more heating elements of the at least one detection chamber; andtransporting the lysate from the waste storage chamber back to the at least one of the one or more extraction chambers after transporting the second set of hybridized magnetic particles to the second detection chamber of the one or more detection chambers9. A diagnostic system comprising:a cartridge comprising a plurality of zones, in fluid communication with each other, comprising:an extraction zone comprising:one or more extraction chambers comprising:Atty. Dkt. No.: 86357089a plurality of sets of magnetic particles, wherein the one or more extraction chambers are configured to hold the plurality of magnetic particles;a waste storage chamber configured to receive a lysate from at least one of the one or more extraction chambers; anda plurality of wash buffer blisters, each wash buffer blister to wash a different set of the plurality of sets of magnetics particles; anda detection zone comprising:a plurality of mixing chambers comprising one or more different reagents;a plurality of detection chambers;a heating element capable of heating the plurality of detection chambers; anda master mix.

10. The diagnostic system of claim 9, further comprising:an instrument to interface with the cartridge, comprising:a lyse system comprising a sonotrode, the lyse system to interface with at least one of the one or more extraction chambers; andat least one of: a plurality of magnetic field generators or a movable magnetic field generator able to interact with the cartridge at one or more detection chambers of the plurality of detection chambers.

11. The diagnostic system of claim 9, wherein the plurality of magnetic particles comprise one or more of: oligonucleotides capable of capturing different nucleic acids of interest, or different types of magnetic particles, each type of magnetic particle having a different oligonucleotide capable of capturing a specific nucleic acid of interest.

12. The diagnostic system of claim 10, wherein a first magnetic field generator is configured to generate a magnetic field to dock the plurality of magnetic particles to an extraction chamber of the one or more extraction chambers; andwherein a second magnetic field generator is configured to capture the plurality of magnetic particles to the heating element.Atty. Dkt. No.: 8635708913. The diagnostic system of claim 9, wherein the one or more different reagents are master mix reagents configured to detect different nucleic acids of interest.

14. The diagnostic system of claim 9, wherein the one or more extraction chambers is a lyse chamber.

15. The diagnostic system of claim 9, wherein the plurality of detection chambers comprise a plurality of amplification chambers to amplify one or more nucleic acids of interest, wherein the one or more nucleic acids of interest are received in the plurality of amplification chambers from the one or more extraction chambers.

16. The diagnostic system of claim 9, wherein the heating element is a foil.

17. A method for serial hybridization, the method comprising:lysing a biological sample into at least one of one or more extraction chambers of a cartridge to release one or more nucleic acids of interest from the biological sample, wherein lysing the biological sample produces a lysate;collecting a first set of hybridized magnetic particles, the first set of magnetic particles hybridized to a first type of nucleic acid of interest by:hybridizing, into the at least one extraction chamber, the first type of nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of a plurality of magnetic particles, the at least one capture oligonucleotide specific to the first type of nucleic acids of interest;holding the first set of hybridized magnetic particles on a side of the extraction chamber;transporting the lysate to a waste storage chamber;suspending the first set of hybridized magnetic particles in the at least one of the one or more extraction chambers with a first wash buffer; andtransporting the first set of hybridized magnetic particles to a first detection chamber of one or more detection chambers; andcollecting, using the lysate, a second set of hybridized magnetic particles, the second set of magnetic particles hybridized to a second type of nucleic acids of interest.

18. The method of claim 17, further comprising:Atty. Dkt. No.: 86357089after collecting the second set of hybridized magnetic particles:transferring a reconstituted first master mix reagent to first detection chamber of the one or more detection chambers;delivering a plurality of amplification reagents unique to the first set of hybridized magnetic particles to the first detection chamber;transferring a reconstituted second master mix reagent to a second detection chamber of the one or more detection chambers, the second set of hybridized magnetic particles to be delivered to the second detection chamber of the one or more detection chambers; anddelivering a plurality of amplification reagents unique to the second set of hybridized magnetic particles to the second detection chamber.

19. The method of claim 17, wherein collecting a set of hybridized magnetic particles further comprises:transporting the lysate from a waste storage chamber R back to the at least one of the one or more extraction chambers after transporting the first set of hybridized magnetic particles to the first detection chamber of the one or more detection chambers, and before collecting the second set of hybridized magnetic particles.

20. The method of claim 17, wherein collecting the second set of hybridized magnetic particles comprises:hybridizing, into the at least one of the one or more extraction chambers, the second nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles, the at least one capture oligonucleotide specific to the second type of nucleic acid of interest;holding the second set of hybridized magnetic particles on a side of the extraction chamber;transporting the lysate to a waste storage chamber;suspending the second set of hybridized magnetic particles in the at least one of the one or more extraction chambers with a second wash buffer, the second wash buffer of a same type as the first wash buffer;transporting the second set of hybridized magnetic particles to a second detection chamber of the one or more detection chambers;Atty. Dkt. No.: 86357089trapping the hybridized plurality of magnetic particles within a threshold distance of one or more heating elements of the at least one detection chamber; andtransporting the lysate from the waste storage chamber back to the at least one of the one or more extraction chambers after transporting the second set of hybridized magnetic particles to the second detection chamber of the one or more detection chambers.

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